Institut für Holztechnologie Dresden • Zellescher Weg 24 • 01217 Dresden • Germany Gütegemeinschaft Paletten e.V. Benrather Schlossallee 2A 40597 Düsseldorf Germany Institut für Holztechnologie Dresden gemeinnützige GmbH Zellescher Weg 24 01217 Dresden Germany Phone: +49 351 4662 334 Fax: +49 351 4662 211 [email protected]www.ihd-dresden.de Dresden, 18/12/2019 Final report - order no. 9218001-A1 This report replaces investigation report no. 9218001 of 28/08/2019, which has been completed and finalised in sections 4.3 to 4.5 and section 6. Client: Order date: Project: Contractor: Person in charge of project: Gütegemeinschaft Paletten e. V. Benrather Schlossallee 2A 40597 Düsseldorf 27/02/2018 Study on the hygienic properties of Euro pallets (load carriers) made from wood, in comparison with Euro pallets made from plastic Institut für Holztechnologie Dresden gemeinnützige GmbH Dipl.-BioI. Katharina Plaschkies Head of Division Biology/Wood Protection This report consists of 38 pages and an appendix comprising 5 pages. The reproduction of parts of this report and appendix is only permitted with the written consent of IHD. The results published in these documents refer exclusively to the examined materials. CEO: Prof. Dr. rer. nat. Steffen Tobisch Dipl.-Kfm. Götz Haake Amtsgericht Dresden HRB 746 VAT ID no.DE 140 30 16 94 Commerzbank AG SWIFT: DRES DE FF 850 IBAN: DE 55 8508 0000 0460 2481 00 Deutsche Bank AG SWIFT: DEUTDEDBCHE IBAN: DE22 8707 0024 0875 ö092 00 r. Wolfram Scheiding SCIENCE BY EXPERTS
For thispurpose, IHD firstcollectedandassessedthe latestknowledgeandtechnology inthe respective fields and examined the hygienic properties of the relevant Euro palletmaterials(softwoodandplastic)foracomparativestudy.Theresultshavebeencompiledinadocumentation that containsbothgeneral findings regarding thehygienicpropertiesofthe examinedmaterials, and specific values determined in experiments and tests of thevariouspalletmaterials.
3.1 ObjectiveTheliteraturesurveywascarriedouttogainanoverviewofthecurrentstateofknowledgeon the hygienic properties of wood and plastics with regard microbial colonisation, thesurvivabilityofmicro-organismsandthetestmethodsforquantitativeassessment.
3.1.1 HygienicpropertiesofwoodandplasticsRequirementsforpalletsusedinfoodindustryThe hygiene requirements for the transport and storage of foods are laid down in theGerman Food Hygiene Ordinance LMHV and the German Food Transport ContainerOrdinanceLMTV.
GS 1Germanyhas definedquality classes (A, B andC) for used EPALpallets. Apart frommechanicalproperties,thissystemalsodefineshygienecharacteristics.WhileclassApalletsmustnotshowanydiscolouration,classBpalletsmightfeaturedarkstains. InclassC,thepalletsmightbemoistonthesurface,andevencontaincontaminantsanddirtas longasthese cannotbe transferredon to the transportedgoods.Pallets showing signsofmouldgrowth inside the material or on its surface are classified as no longer usable. For laypersons, it isnotalwayseasy todeterminewhetherdiscolouration is causednaturallybylightandoxygen,bydirtorbymicrobialcolonisation.
For international trade, the requirements for packaging and transport equipment madefrom wood are defined in various standards, such as ISPM 15. These standards aim atpreventingthespreadingofharmfulorganismsandthusfocusonphytosanitarymeasuresrather than hygienemeasures in the narrow sense, as they do not specifically dealwithhumanoranimalpathogens.
ConditionspromotingmicrobialcolonisationinmaterialsClimateconditionsplayakeyrole,wherebymoistureavailabilityhasbeenidentifiedasthemain limiting factor (Hankammer and Lorenz 2003).Most bacteria only form colonies onsurfaces,iftherelativeairhumidityis98-100%,correspondingtoanAwvalueof0.98-1.00.The Aw value is themeasuring value forwater activity, i.e. thewater freely available tomicro-organismsinthesubstrate.Halophilicandhalotolerant1speciesarehowevershowinggrowthathumiditylevelsofaround60%.Mouldgrowthstartsatapprox.70%relativeairhumidity,wherebymost species only thrive at an air humidity above 85% (Mack 2000).Another limiting factor is temperature.Bothbacteriaand fungigrowwithinanextremelybroadtemperaturerangefromafewdegreesCelsiusabovezerotoaround40°C,andsomespecieseventoleratetemperaturesoutsidethisrange.Thenaturalenvironmentcontainsahugevarietyofmicro-organismsabletocolonisematerials,providedtheclimateconditionsare right. Their growth can however be prevented by choosing materials with specificproperties or special treatment. Growth is for instance hampered by naturally occurringbiocidal substances, pH values outside the range of 2 to 11, protective impregnation ortreatmentwithbiocidalproducts.
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1preferortoleratehighsaltconcentrations
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When micro-organisms grow on the surface of materials, they normally form biofilmscontaining various species. Inside these biofilms, the organisms are protected by a polymermatrixagainstadverseinfluencessuchasdesiccation,UVlight,extremepHortoxicsubstances(Hall-Stoodleyetal.2004).
As dirt on the surface of amaterial aswell as air-borne dirt such as dust, grease and sweatprovidearichsourceoffoodformicro-organisms,suchbiofilmsevenformon inertmaterialsincludingglassandmetal.Withregardtopallets,dirtdepositscanoccurinmanyindustries,andespecially in the food-processing sector, so that there is always a risk of organic deposits onpallets,iftheyhavepreviouslybeenincontactwithunpackedgoods.
MicrobialgrowthonplasticsThe ambient conditions described above also apply to microbial growth on plastic surfaces.Mosthygienepalletsaremadefrompolyethylene(PE)orpolypropylene(PP).Theseareorganicpolymerssynthesisedfromethyleneorpropylenerespectively, twomaterialsareclassifiedasnotreadilybiodegradable.Low-molecularpolyethylenecanhoweverbecolonisedbybacteria(Jen-Hou and Schwartz 1961), and partly degraded bymoulds that produce special enzymes(peroxidases) (Iiyoshi et al. 1998). Latest studies on microplastic waste in the oceans showhoweverthatplasticsarenotdegradedinnature(OverbeckmannandLabrenz2019).
Plasticsareprimarilyusedforapplicationswheremicro-organismgrowthwouldhaveaseriousnegativeimpactonhealth,asisthecaseindrinkingwaterdistributionsystemsandthefieldofmedicine. In Germany, drinking water pipes must be made from plastics that meet therequirements laid down in the W270 standard of the German Technical and ScientificAssociationforGasandWater(DVGW),toensurethatnobiofilmscanformovertimeonthesurfacesexposedtodrinkingwater(Kötzschetal.2016).Thisdoeshowevernotmeanthatthematerialcannotnotbecolonisedbymicro-organismsunderdifferentconditions(Kötzschetal.2017). There are many plastics that meet the stringent requirements of W270, andpolyethylene is for instance widely used for drinking water pipes(https://www.baunetzwissen.de).
In the field ofmedicine where particularly high hygiene standardsmust bemet, the use ofplastics iswidespread.Toensurepatientsafety,devices,consumablesandimplantsaremadefromtop-gradesyntheticmaterials,wherebybiocompatibilityandeasycleaningaretwoofthekey considerations. Medical-grade plastics often contain anti-microbial components such asmetalsaltsorsliverions(https://medlexi.de).
Mouldattackisacommonprobleminwoodwithahighmoisturecontent.Inoutdoorareas,moulds cause primarily unsightly discolouration. Indoors, mould attacks pose a hygieneproblem,as these fungi canbeharmful tohumanhealth, for instanceby causingallergicreactions. Theactualhealth riskdependshoweveron the typeof themould, the speciesandtheexposuretoit.Somepeoplearemoredisposedtoadversereactionstomouldsthanothers.
If the conditions are right, virtually all solid wood and other timber-basedmaterials canbecomecolonisedbymoulds,exceptthosewithanextremelyhighpHof>11,asisfoundforinstanceincement-bondedparticleboards.Aspalletsarenormallymadefromspruceorpine, their surfacesprovidean ideal substrate formouldgrowth,wherebypine is slightlylesssusceptibletomouldattack(Scheidingetal.2003).
Aspecialtypeoffungalattackaffectingwoodiscausedbyblue-stainfungiwhich,togetherwithmoulds,belongtothephylumofAscomycota.Thedark-brownfungalhyphaeextenddeep into the wood. The blue colour is the result of optical refraction on these brownfilamentous structures. Blue-stain fungi are commonly found in softwood species, whilehardwoodssuchasmapleorbeechare lesssusceptible toattack.Blue-stain fungi liveoffsubstances contained in the parenchyma cells of sapwood and do digest structuralsubstancessuchas ligninandcellulose,so that thestructural strengthof thewood isnotaffectedbyanattack.Bluestainalsoposesnohealthhazardtohumans(UBA2019),sothattheuseofthepalletsaffectedbythistypeoffungiisunproblematic.
Inbuildingconstruction,fungalattackintimberisamajorconcern,whilebacterialattackisa minor issue. Although bacteria also contribute to the digestion of wood, they need amuch highermoisture content thanmoulds to actually cause problems. In humid indoorareas,mouldattack thereforealwaysoccursbeforebacterialgrowth,and isgenerally theprincipalcauseofwooddecay.
Where the spreadingofpathogensand food-spoilinggermsmustbeprevented,bacterialgrowthonsurfacesisobviouslyamajorconcern.Asaresult,therearemanystudiesontheanti-bacterialpropertiesofwoodforuseinfoodprocessingandstorage.Otherstudieslookattheuseofwoodinhospitalinteriors.Thefindingsoftheliteraturesurveyarecompiledinthenextchapterwhereweexamineandcompareanumberofhygienicpropertiesofwoodandplastics.
ComparisonofthehygienicpropertiesofwoodandplasticsAnumberofolder studiesexaminewhetherwood ismorehygienic thanplastic. Inmostcases, these comparisons refer todirect (ason choppingboards)or indirect contactwithfoodstuffs (Ak et al. 1994, Weiker et al. 1997, Gehrig et al. 2002, Prechter et al. 2002,Schönwälderet al. 2002,MühlbauerandMilenovic2012,Kleinerand Lampe2014, LückeandSkowyrska2015).
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Other studies assess the suitability of wood as an interior building material where highhygiene standardsneed tobemet, such as in hospitals and clinics (Strehlein et al. 2004,Schusteretal. 2006). Therearealso somestudies that lookat thehygienicpropertiesofpackagingmaterialsSteinkampandWilms2000,HPE2014,Milling2005-1).
Using a range of different methods, nearly all studies conclude that wood has certainantibacterial properties, be it due to its structureor its chemical composition.Wood is aporousmaterialwithaverylargeinsidesurfacearea.Woodishygroscopic,whichreducestheavailabilityofwaterforbacteria.Dependingontheactualtreespecies,woodcontainsantimicrobialsubstances(StinglandHansmann2006).
AcomprehensiveoverviewofthehygienicpropertiesofwoodwascompiledbyAviatetal.(2016)whoreviewed86publicationsinordertodeterminewhetherdirectcontactbetweenwoodand foodstuffs is safe. Theauthorsdiscuss theantimicrobial propertiesofwoodaswell as the testing methods used in the reviewed studies. They conclude that wood issuitable fordirect foodcontact,as its roughsurfaceandporousstructureoftengenerateunfavourable conditions for micro-organisms and/or bind or trap them in the material.These physical characteristics of wood, rather than any potentially antimicrobialconstituents, have been found to be themain reason for the antibacterial properties ofwood (Lukowsky 1994). Compounds found in wood that have potentially antimicrobialproperties belong to the various groups including phenols, lignans, tannins, stilbenes,flavonoidsandterpenoids(Pearce1996,MoureyandCanillac2002).
Ofspecial interestherearehygienepalletsmadefrompineheartwood.Steinkamp(2004)performedlaboratoryandfieldtestswithrelevantgermsfrommeatandanimalproduction,and from hospital environments. The results show that hygiene pallets made from pineheartwoodhad clear antibacterial properties and thusoutperformplastic pallets. FurtherproofoftheantibacterialactivityofpineheartwoodcanbefoundinMillingetal.(2005-2)andRipolles-Avilaetal.(2019)whoexamineditsuseinthetransportoffreshfish.
Detectionmethods
There is currently no standardised method to determine which bacteria survive underspecificconditions.AgardiffusionplatetestsaccordingtoDINENISO8462orDINEN11043are not useful, as they only determine whether the wood excretes antimicrobialconstituents. In the context of food safety, such excretions are however problematic, asarticle 3 of Regulation (EC) No. 1935/2004 prohibits the transfer of antimicrobialconstituentsfromthepackagingtofoodstuffs,asthesesubstancescanadulteratethefood.
For practice-oriented research projects, methods where the material is inoculated withbacteria,whicharethentransferredtoaculturemediumfor incubationhavebeenfoundmoreusefultodeterminethesurvivalofbacteriaoncertainmaterials.
Withsuchmethods,samplestakenfromthesurfaceofthematerialunderinvestigationaretransferredwith a punch under pressure to a solid culture substrate (Gehrig et al. 2002,Fürst2007,Kavian-Jahromi2015),whichhowevermeansthatbacteria insidethematerialarenotcomprisedinthesamples.Moresuitableformostpurposesrinsingmethodswherethesurvivalrateofthebacteriaisdeterminedbyrinsingthemoffthematerialfollowedbytransfertotheculturemedium.Withrinsingmethods,thereishoweversomeuncertainty,asitisnotclearwhetherallbacteriaareremovedfromthematerialintheprocess.Whilesimplerinsingissufficienttodetachallbacteriafromsmoothplasticsurfaces,therecoveryratefromporousorroughwoodsurfaces isrelativelysmall (Carpentier1997),butcanbeincreasedbyultrasonictreatment(LeBayonetal.2010)orbrushing(Marianietal.2007).Thehighest recovery rate isachievedwith scraping,which shows thatbacteriapenetratethewoodandbecomefirmlyattachedtoit(Ismailetal.2014).
Themethoddeveloped todetermine theantibacterial activity onplastics andothernon-poroussurfaces(ISOCD22196)canbeappliedwithoutmodificationstotheexaminationofplastic pallets. It is however not suitable to test untreatedwood, which is by its natureporous,sothatthemethodalwaysreturnsamuchlowerbacteriacountasonlyafractionofthemicro-organismsarerinsedfromthematerial.
Ak No, Cliver Do, Kaspar Cw (1994) Cutting Boards of Plastic and Wood ContaminatedExperimentallywithBacteria. JournalofFoodProtection: January1994,Vol.57,No.1,pp.16-22.
FürstD (2007) VergleichendeUntersuchung der antimikrobiellenWirksamkeit von siebenverschiedenen Hölzern. Dissertation. Albert-Ludwigs-Universität Freiburg i. Br.,MedizinischeFakultät.
Jen-Hou L, Schwartz A (1961) Verhalten von Bakteriengemischen gegenüber PolyethylenverschiedenenmittlerenMolekulargewichts.KunststoffeNr.51,pp.317-320.
Kavian-JahromiK(2015)Comparisonoftheantibacterialeffectsofsapwoodandheartwoodof the larch tree focusing on the use in hygiene sensitive areas Eur. J. Wood WoodProducts73(6),841-844(2015)
Kleiner U, Lampe U (2014) Vergleichsuntersuchungen zum Hygienestatus von Holz- undKunststoffschneidbrettern im Labormodell. Rundschau für Fleischhygiene undLebensmittelüberwachung66(9),pp.319—322.
Kötzsch S, Rölli F, Sigrist R, Hammes F (2016) Kunststoffe in Kontakt mit Trinkwasser.HygienetestimVergleich.Aqua&Gas(12),pp.43-52
Kötzsch S, Rölli F, Sigrist R, Hammes F (2017) Trinkwasserqualität in Gebäuden.Synthesebericht:KTI-Projekt"MaterialieninKontaktmitTrinkwasser".Aqua&Gas(10),pp.74-78
LeBayonI,CallotH,KutnikM,DenisC,Revol-JunellesA-M,MilliéreJ-B,GiraudM,GabilléM, Passédat N (2010) Development of microbiological test methods for the woodenpackagingof foodstuffs. IRG/WP10-20453.Biarritz, France.The InternationalResearchGrouponWoodProtection.
LückeF,SkowyrskaA(2015)Hygienicaspectsofusingwoodenandplasticcuttingboards,assessed in laboratoryand small gastronomyunits. J.Verbr. Lebensm.10 (4),pp.317-322.DOI:10.1007/s00003-015-0949-5.
Biofilmecologyofwoodenshelvesused in ripening theFrench rawmilk smearcheeseReblochon de Savoie. Journal of dairy science 90 (4), pp. 1653-1661. DOI:10.3168/jds.2006-190.
Milling A, Kehr R, Wulf A, Smalla K (2005-2): Survival of bacteria on wood and plasticparticles:Dependenceonwoodspeciesandenvironmentalconditions.Holzforschung59(1),p.16.
HolzRohWerkst63(6),pp.463-472.Mourey A, Canillac N (2002) Anti-Listeria monocytogenes activity of essential oils
components of conifers. Food Control 13 (4-5), pp. 289-292. DOI: 10.1016/S0956-7135(02)00026-9.
Mühlbauer M, Milenovic N (2012) Hygienische Eigenschaften von Holz im Vergleich zuKunststoff. Diplomarbeit. Höhere Technische Bundes, Lehr- und VersuchsanstaltMödling.AbteilungHolztechnik.
Oberbeckmann S, Labrenz M (2019): Marine microbial assemblages on microplastics:diversity, adaptation, and role in degradation. Annu Rev Mar Sci;https://doi.org/10.1146/annurev-marine-010419-010633.
Ripolles-AvilaC,HascoëtAS,Rios-CastilloAG,Rodriguez-JerezJJ(2019)Hygienicpropertiesexhibited by single-use wood and plastic packaging on themicrobial stability for fish.LWT113,p.108309.
Scheiding W, Kruse K, Plaschkies K, Weiss B (2003) Untersuchungen zum VerhaltenausgewählterBau-undHolzwerkstoffegegenüberSchimmelpilzen.AbschlussberichtzumBMWi-ForschungsprojektReg.-Nr.39/01
Schönwälder A, Kehr R, Wulf A, Smalla K (2002) Wooden boards affecting the survival ofbacteria?
HolzalsRoh-undWerkstoff60(4),pp.249-257.SchusterA,Schmidt-EisenlohrE,DaschnerF(2006)Wiehygienischundsinnvoll istHolz in
Patientenzimmern?In:Krankenhaushygiene+Inf.verh.(28vol.4),pp.131-137.Steinkamp H,Wilms H (2000) Untersuchungen zur Einführung von Hygiene-Paletten aus
Holz zum Einsatz in der Lebensmittelindustrie. Abschlussbericht zum FuE-Vorhaben,Quakenbrück.DeutschesInstitutfürLebensmitteltechnike.V.
Steinkamp H (2004) Antibakterielle Wirkung von Holz - Einsatz als Filter- undAbsorptionsmaterial.NaRo.Net-Holzforum.Osnabrück,19/04/2004.
Stingl R, Hansmann C (2006) Holz und Hygiene. Antibakterielle Eigenschaften vonMaterialien.proHolzZuschnitt22.
Strehlein M (2004) Nutzung von Holz im Krankenhaus unbedenklich. Studie weistantimikrobielleWirkungvonKiefernkernholzgegenErregervonKrankenhausinfektionennach.Holzzentralblatt(71),pp.951-952.
WelkerC,FaiolaN,DavisS,MaffatoreI,BattCa(1997)BacterialRetentionandCleanabilityof Plastic and Wood Cutting Boards with Commercial Food Service MaintenancePractices.JournalofFoodProtection60(4),pp.407-413.
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Sourcesontheinternet
Bundesverband HPE e.V. (2014) Keimabtötende Eigenschaften von Holzpackmitteln.Available online https://www.neue-verpackung.de/26569/keimabtoetende-eigenschaften-von-hoIzpackmitteIn/Accessed29/07/2019.
According to the objectives of this study, pallets from various manufacturers, users andprocessors in a range of industries (e.g. food processing, animal feed production,agriculture)weremicrobiologicallyexaminedandassessed.Theclientwasresponsible fortheselection,procurementandprovisionofthematerialforexamination.
The following wood pallets were delivered by Collico Verpackungslogistik und ServiceGmbHon29/11/2018tothelab:
The supplied pallets had been in use. The stamps (if applied) indicate their origin; thecontractor had no further information regarding their previous use or ownership. Thestampdetailsarelistedintable1.
All pallets conform to theEPA standardand consistof 13boards and9blockseach. Thedeterminationof thewoodspecieswasnotpartof thescopeof this study.Apreliminaryassessmentof somerandomsamples indicateshowever thatmostof thepalletelementsaremade fromsoftwood,with fewhardwoodcomponents. Theblocksare solidwoodorwoodchipblocks(Presspan);somepalletscontainacombinationofthesetwoblocktypes.Fordetails,seesection4.4,table2.
Pack1.1E1 x DE-SN1 497027 021-3-03 EPALE2 illegible EPALE3 x illegible illegible ...-6-03 UICE4 x illegible illegible EPALE5 x DK 8015 GC-148-5-0 EPALE6 x illegible illegible 024-4-07 EPALE7 x DE-RP illegible illegible EPALE8 illegible EPALE9 x BY 009 010-2-02 EPALE10 x H illegible 604-2-05 MAV
Pack1.2
E11 x SK 2291 2156A27-7-11 UICE12 x SK 2291 2156A27-7-08 UICE13 x PI 18153 152-7-01 EPALE14 x LV 012 008-4-03 EPALE15 x SK-3231 2291 2156A27-8-01 UICE16 x DE-HE illegible illegible EPALE17 x RO-CV illegible illegible EPALE18 x BE 99012 012-3-09 EPALE19 x GB FC-035 05-3 EPALE20 x BA illegible 003-6-05 EPAL
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Pack1.3E21 x DE-HE 493027 003-0-12 EPALE22 x DE-SN1 497027 021-3-07 EPALE23 x SK 2291 2156A27-5-09 UICE24 x illegible illegible 004-5-11 EPALE25 x SK 2291 2156A27-7-08 UICE26 x SK 2291 2156A27-7-95 UICE27 x PL 02-387 181-8-01 EPALE28 x DE-SN1 497027 021-5-05 EPALE29 x ROCV illegible 004-7-07 EPALE30 x DE-SN1 497027 021-6-12 EPAL
4.3 PlasticpalletsforexaminationAdeliveryoffiveplasticpalletswasreceivedon28/11/2019.Accordingtotheclient,thesehad been previously used bymeat-processing companies. The contractor had no furtherinformationregardingtheirprevioushistory.ThepalletsborenolabelsorstampsandwerenumberedbythecontractorK1toK5.
ThesamplesweretakenbypressingspecialcontactPetridisheswithasolidculturemediumontothematerialinordertocollectmicro-organismsfromthesurfaceofthematerial.Thefollowing culturemediumswere used: DG18 agar for xerophilicmoulds andmalt extractagar (MEA) as a universal medium for moulds and bacteria. The Petri dishes were thenincubatedforoneweekinanincubatorat25°C.Subsequently,themicro-organismcolonieswere counted and the species were determined based on micromorphological andmacromorphologicalcharacteristics.
Fortheexaminationoftheplasticpallets,swabsamplesweretakenatfivepointsfromanareaof20cm2each.Thesesamplesweretakenfrombothvisiblycontaminatedandfrominconspicuousareas. The swab sampleswereeluded in saline solution (0.9%) containingTween80,andthentransferredwithaspiralplatertovariousculturemediaforincubation.Here,maltextractagar,DG18agarandplatecountagarwereused.Theincubatedcolonieswerecountedandthespeciesweredeterminedbasedonmorphologicalandbiochemicalcharacteristics.
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Quantitative and qualitative determination of the microbial surface contamination ofwoodpalletswiththesuspensionmethod,usingwoodcross-sectionsFor this examination, twobore coreswere taken fromeachpallet. Thebore coreshad adiameterof10mmandweretakenfromhorizontaldeckboardsinareaswheretherewasnovisibledirtormould.SamplingwasperformedaccordingtoDINISO16000-21:21014-054.Theborecoresweretransferredinstantlyintoa10mlphosphatebuffersolution.Tobringall germs into solution, the sampleswereprocessedonanorbital shaker for15minutes.Theeluatedilutionstageswerethentransferredwithaspiralplatertotheculturemediumforincubationandsubsequentgermcount.
Mostpallets showsomegreyedareasandblue-stainingof thewood,whicharehowevernot deemed quality defects. 18 pallets were contaminated to some degree, often byfootprints or black traces from forklift arms. The pallets of pack 1.1 were contaminatedmorethantheothers,showingvisiblemouldattackintheformofcolouredfilmsatvariouspointsof thepallets. In total, 14pallets showedmouldattack to adegree that renderedthemnolongersuitableforuseaccordingtotheEPALqualitycriteria.
Alargenumberofdifferentfungicouldbeidentifiedinthecontactsamplesontheculturemedia.ThepredominantmouldspeciesbelongedtothegenusofPenicillium,Cladosporium,Aspergillus and Trichoderma. The most common blue-stain species was Aureobasidiumpullulans. All species of fungi identified in this study are also commonly found onwoodused in other areas, in particular building construction. In addition, someof the samplesalsocontainedbacteriaandyeasts.
Themicroscopicexaminationoftheborecoresrevealedsignificantdifferenceswithregardtocolonisation(seetable5).Inthemajorityofthesamples(20pallets),themicro-organismconcentrationpervolumewasbelowthedetectionlimit,reflectinganormalmicrobialload.The concentrations determined from pallets of pack 1.1. were considerably higher thanthosefromotherpallets.Here,fivepalletstoppedthetablewith>100,000CFU/cm3.
Forthe interpretationofthesefindings,onemusttake intoaccountthatthepalletsweredeliveredinpackswheretheywereinclosecontactwitheachother,facilitatingthetransferof fungal spores. The fungi cultivated fromcontact samplesare thereforenotnecessarilythe speciespredominanton the respectivepallets.On theotherhand, the findings showthat therewere viablemould structures on allmaterials, ready to germinate and spreadquickly,iftheconditionswereright.
4.5.2 PlasticpalletsAllplasticpalletsshowedsignsofwearintheformofscratchesontheirsurfaces.Someofthemwere clearly contaminated, for instancebydark stains, orwithbloodandmeat residue. Thegerm counts of the plastic surfaces determined by swab sampling are compiled in table 6.Picturesoftheincubatedsamplesonculturemediaareincludedinthesectionbelow.
PalletK1Thispalletwasquiteheavilycontaminatedbydriedmeatresidueandbloodstains.Therewasno clearly identifiable microbial attack, and the microbial load became only apparent afterincubation. The predominant micro-organisms were yeasts (including Rhodotorulamucilaginosa) and bacteria. In samples containing dried meat residue, primarily coliformbacteria, other enterobacteria including Escherichia coli, as well as gram-positive cocci wereidentified.Samplestakenfromareasfreeofvisibleresidueshowedlowgermcounts(K1.5).
PalletK2ThispalletwassimilarinappearancetopalletK1,showingrelativelyheavycontaminationinthe form of dried meat residue and blood stains. The microbial load was howeversignificantlylowerthanonpalletK1.Thepredominantgermswhereyeasts,gram-negativebacteria includingenterobacteria,and fewmoulds.Thepicturesbelowshowthecoloniesonculturemediaproducedfromthefiverandomsamples.
AreaK2.1:driedmeatjuiceontopside
MEA
AreaK2.2:unobtrusiveareaontopside
MEA
AreaK2.3:driedmeatjuiceontopside
MEA
AreaK2.4:driedmeatjuiceinrecess
MEA
DG18DG18DG18DG18
PCA
PCA
PCA
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AreaK2.5:driedmeatjuiceinbottomarea
MEA DG18
PalletK3Thispallet showedsignsofnormalwearandgenerally lowcontamination,apart fromsomewet,transparent stains. Despite these minor stains, there was considerable microbial contamination,dominated by yeasts and gram-negative as well as gram-positive bacteria. The yeast/bacteriaconcentrationwasparticularlyhighinthesampledwetarea(K3.1).Samplesfromunobtrusiveareasalsocontainedmoulds(Aureobasidiumpullulans,Cladosporiumsp.,Alternariasp.).
AreaK3.1:wetareaontopside
MEA
AreaK3.2:darkstainontopside
MEA
AreaK3.3:unobtrusiveareainrecess
MEA
DG18
DG18
DG18
PCA
PCA
PCA
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AreaK3.4:slightlydirtyareaonbottomside
MEA
AreaK3.5:slightlydirtyareainrecess
DG18 PCA
MEA DG18 PCA
PalletK4Thispallet showedsignsofnormalwear,wasgenerallydirtyandpartlywet.Again, therewas considerable microbial contamination by yeasts and bacteria, with few moulds(Paecilomyces variotii). The yeast/bacteria concentration was particularly high in thesampledwetareas(K4.1andK4.3).
In this work package, a number of pallet materials (wood and plastic) were examined todeterminethelikelihoodofmicrobialcolonisation,andthesurvivabilityofbacteria.
An initial investigationbasedonrandomsamples revealedthatnotallwoodwasspruce,andthatthepalletsalsocontainedpinewood.TheEuropalletswerethereforenotincludedinthesubsequentexamination.
To replace these pallets, the client contracted Treyer PalettenGmbH in Peterstal to send usboardsmadefromspruceandpine,whichweredeliveredon31/05/2019.Thepinewoodhadbeenseparatedbytheclientintosapwoodandheartwood.
TestprincipleThetestsampleswereinoculatedwithaknownbacterialsuspension,andthecultureswereincubatedforaperiodbetween18to24hoursat36°Cinahumidchamber.Subsequently,thesurvivalratewasdeterminedwiththeluminescencemethod.Inordertodeterminetheeffect of cleaning, some of the cultures were cleaned, using various methods, afterincubationandpriortodeterminingthesurvivalrate.
RemovalofbacteriabyrinsingafterincubationThe samples were placed on glass beads in cell culture plates, with the inoculated sidedown. After addition of 6 ml of phosphate-buffered saline solution, the plates wereprocessedonanorbitalshakerat250rpmfor15minutes(fig.2and3.).Subsequently,thesurvivabilityofthebacteriaintheeluatewasdeterminedwiththeluminescencemethod.
The luminescencemethodmakes use of the fact that all living cells have the capacity toproduceadenosinetriphosphate(ATP)inordertostoreandtransferenergywithinthecell.The presence of ATP can be detected with the luciferin-luciferase reaction, where ATPprovides the energy to convert the luciferin into light, using the luciferase enzyme. Theamount of light emitted in the process is proportional to the available ATP, so that theluminescencecanbeusedtodeterminethesurvivabilityofmicro-organismsandthusthelivegermcount.ThemethodisthereforealsoknownasATPluminescencetest.
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For our test,we used the "bactiter glo" test system fromPromega. For testing, 50 μl ofeluatewasmixedwith50μlofthedetectionreagentin96-wellmicro-titrationplates.Afteranincubationperiodof5minutes,theluminescencewasmeasured.
5.4 FindingsThemeasuredluminescencesignal isanindicatorofbacterialactivity.Theexaminationofthe bacteria suspensions used for inoculation showed a linear relationship between theluminescence signal (RLU) and the number of live cells (fig. 4), so that the RLU could beusedtocalculatetheactualbacterialcount.
Fig.4:Calibrationcurve:RelationshipbetweenbacterialcountandluminescencesignalThe results of the luminescence measurements are compiled in table 8 and thechartsinfig.5.
After incubation over 24 hours, all wood samples showed similar results. The activity ofEscherichia coli was significantly reduced in all woods, irrespective of the actual testversion,while it increasedonnearlyallplasticpallets.Theonlyexceptionshereweretheresultsoftestversion3(additionofnutrientsolution),whichshowedareductioninactivity.
The results for Staphylococcus aureus were similar, with the exception of the pineheartwood samples where no significant change in activity could be measured afterincubation.Incontrast,theluminescencesignalsmeasuredwiththeplasticpalletsampleswere significantly higher after incubation than before. On the rough plastic surface,Staphylococcusaureusgrewmuchfasterthanonthesmoothsurface.
ExploratorystudyregardingtheadhesionofbacteriaonthetestedmaterialsUsing additional test samples and Escherichia coli, a number of tests were performed todeterminetowhichextendthechosenshakingmethodwasabletodetachthebacteria fromthematerial.Forthispurpose,samplestakenfrompinesapwoodandfromplasticpalletsweredried after inoculation or shaking respectively, and then examined in a scanning electronmicroscope(SEM).Bacteriawereonlyclearlyvisibleonthe inoculatedsamples.This indicatesthattherinsingprocessusedintheabovetestswaseffective,andthatonlysmallamountsofbacteria remained attached to the material, especially to the wood. Figures 6 to 9 showrepresentativeimagesofthefindings.
Fig.8:Pinesapwoodafterinoculationand Fig. 9: Pine sapwood after inoculation,incubationfor2hours: incubation for 2 hours and shaking: nobacteriaclearlyvisible(SEM) bacteriavisible(SEM)
Inthevarioustests,lessbacteriasurvivedonwoodthanonplastic.Adistinctantibacterialactivityofpineheartwood,assuggestedinthesurveyedliterature,couldnotbeconfirmed,as the activity in some samples was lower than that of spruce and pine sapwood. It ishoweverpossiblethatthetestedsampleswerenotactuallyofheartwood.
Woodhassomedisadvantageswithregardtocleaning.Asitisaporousmaterial,itcannotbecleanedaseasilyasthesmoothplasticsurfaces.Itmustbeassumedthatcontaminants(and in particular organic substances) become firmly attached to the wood surface,providingapotential feedingground forbacteriaandmoulds.The tests showedhoweverthat simple cleaningwithwater is highly effective and significantly reduces the bacterialcountonbothplasticandwoodsurfaces.
Theantimicrobialpropertiesofplasticpalletsaregreatlyaffectedbyscratchesandroughareas on the surface, which are probably unavoidablewhen pallets are re-usedmultipletimes. The bacterial counts on rough plastic surfaceswere higher than those on smoothsurfaces.
Microbialgrowthonusedmaterial
Thewoodandplasticpalletsexaminedinworkpackage2showedsignificantsignsofwear,as well as high microbial loads in certain areas. On the wood pallets, moulds were thepredominant micro-organisms, with few bacteria and yeasts. On the plastic pallets,significantlymoreyeastsandbacteriapronetospoilfoodwerefound.Thelivegermcountonthesurfaceofthewoodpalletswasbetween<10CFU/cm2and>30CPU/cm2.Incross-sections,thelivegermcountwasbetween2x102CFU/cm3and5x105CFU/cm3.Onplasticpallets,thelivegermcountonthesurfacewasbetween<10CPU/cm2and105CFU/cm2.
Acomparisonoftheabsolutevaluesofthetwogroupsofmaterialsisnotpossible,duetothedifferencesinthemethodsusedfortheexaminationofthetwotypesofpallets,andthefact that thehistoryof the individualpalletswasnot known.The findings showhoweverthatmicrobialattack isacommonandserious issueinthepracticaluseofpallets.Onthewood pallets,microbial attack, in particular bymoulds, was often clearly visible, so thataffectedpalletstendtobeseparatedanddisposedof.Incontrast,themicrobialgrowthsontheplasticpalletsweexaminedwerenotidentifiablewiththenakedeye,althoughourtestsshowedthatthereweresignificantgermconcentrations.
Laboratorytestsshowthat, ingeneral, thesurvivabilityofbacteriaonwood is lowerthanonplastic.Itisthereforesafetoconcludethatwoodpalletsaresuitableforuseinfoodprocessingand transport where hygiene is of great importance. However, such use requires strictadherence to the hygiene regulations and standards that apply to the production, transportandstorageoffoodstuff,includingcontinuouscontrolofthepalletqualityandregularcleaning,whicharerequirementsthatapplyofcoursealsotoplasticpallets.Topreventmicrobialattack,thematerialmustbeprotectedagainsthumidityanddirt,andregularlycleaned.
