1.01 Overview of Research Needs, Future and Potential Applications of High- Pressure Processing RobertSevenich,Cornelia Rauh,andDietrich Knorr,Department ofFoodBiotechnology andFoodProcess Engineering, TechnischeUniversität Berlin, Berlin, Germany ©2021ElsevierInc.Allrightsreserved. 1.01.1 Introduction 1 1.01.2 VegetativeMicroorganisms,Spores,Viruses,Parasites,BacteriophagesandNematodes 2 1.01.2.1 VegetativeMicroorganisms 2 1.01.2.2 Spores 3 1.01.2.3 Viruses,Bacteriophages,ParasitesandNematodes 4 1.01.3 ChemicalReactions:InfluenceonAllergens,Toxins(FoodBorneandAgriculturallyBased) 5 1.01.3.1 Allergens 5 1.01.3.2 Toxins 6 1.01.3.2.1 FoodProcessingContaminants 6 1.01.3.2.2 Aflatoxins,PesticidesandHerbicides 7 1.01.4 Process-Structure-Relationship 7 1.01.4.1 Starch 8 1.01.4.2 Pectin 8 1.01.4.3 Proteins 9 1.01.5 PackagingMaterial 10 1.01.5.1 Polylactides(PLA) 11 1.01.6 DataReportingandExperimentalDesign 11 1.01.7 Conclusion 11 References 14 1.01.1 Introduction Today,theconsumers’demandforhighqualitysafefoodsrequiresthedevelopmentandapplicationofemergingprocessingtech- nologiesforthegentlepasteurizationandsterilizationoffoods.Therefore,thefoodindustryislookingfornewwaystoproduce safe,healthyandstablefoods.Onewaytomeetthisaimisapplyinghighpressure,intheorderof600MPa,tofoods,whichis referredtointheliteratureascoldpasteurization(Matseretal.,2004;Knoerzeretal.,2010;Mújica-Pazetal.,2011).Currently, high-pressureprocessing(HPP)inthefoodindustryissolelyusedforpasteurizationpurposes.Thetrendofusinghighpressure as a technology for pasteurization of different kinds of foods, e.g., juices, ham, sauces and seafood is a growing sector in the foodindustrysincethe1990’s(HoganandKelly,2005).Pasteurizationoffoodswithhighpressure(HP)iswellestablishedin the food industry. In 2018, 500 industrial-scale high pressure systems were in use worldwide, producing approx. Four million metrictonsannuallyofpressurizedfoods.Thereisstillaneedforresearchinthisfield,e.g.,consideringtheimpactonfoodstruc- ture,foodquality,microorganisms,enzymeactivity,andnutrients.Furthermore,thereisahighacceptanceofpressurizedfoodson theconsumersideduetothecleanlabelandhealthpromotingattributesthetechnologyoffers(Olsenetal.,2010),withoutwhich industrialapplicationwouldbelessattractive.Despitethesteadilyincreasingcommercialproductionofhighpressuretreatedfood, someimportantscientificandtechnologicalquestions,aswellassomepotentialotherapplicationsofHPParestillunresolved.One oftheseissuesistheimpactofdifferentintrinsicandextrinsicfactorsontheinactivationmechanismsofvegetativebacteriaand bacterialsporesunderpressure.Tounraveltheimpactofthedifferentpressureandtemperaturecombinationsonapossiblecell deathorrecovery,detailedanalysesaboutthephysiologicalstateofthecellsandhowtheyareinfluencedbydifferentfoodcompo- nentsareneeded.AccordingtoLeChatelier’sprinciple,inasystemfacingashiftofequilibrium,allcellularcomponentsareaffected byhighpressure,includingthecellmembraneanditsmembraneproteins,enzymesandribosomesaswellastheentirecellmetab- olism(WinterandJeworrek,2009;Georgetetal.,2015).Ingeneral,prokaryoticcellsshowahigherresistancetowardpressurethan eukaryoticcells.Yeastsandmoldsareingeneralmorepressuresensitive,althoughascosporesofsomemoldssuchasByssochlamys andTalaromycescanbeverypressureresistant(Smelt,1998;Considineetal.,2008;Georgetetal.,2015).Withinprokaryotes,gram positivemicroorganismssuchasBacillus,Listeria,StaphylococcusandClostridiumhaveathickerpeptidoglycanlayerandaretherefore morepressureresistantthangram-negativemicroorganisms(Smelt,1998;Considineetal.,2008;Dumayetal.,2010).Themech- anismsleadingtocelldeathhavebeeninvestigatedinseveralbacterialspecies(Huangetal.,2014).However,theparticularevents leadingtoinactivationarenotwellunderstood(Cheftel,1995;BuckowandHeinz,2008;Klotzetal.,2010).Highpressurebetween 300and800MPaatambienttemperaturescanleadtotheunfoldinganddenaturationofimportantcellenzymesandproteinsin vegetativemicroorganisms(Rastogietal.,2007;Knorretal.,2011a,b),butthespecificpressureeffectsonmicroorganismsaremore InnovativeFoodProcessingTechnologies,Volume1 https://doi.org/10.1016/B978-0-08-100596-5.22991-0 1 2 Overview ofResearch Needs,Future andPotential ApplicationsofHigh-Pressure Processing complexandseveraldifferentmechanismsleadingtocelldeathcaninteractwhenhighpressuresareapplied.Primarily,pressureat asufficientlyhighlevel,caninduceenzymeinactivation,membraneproteinsdenaturationandcellmembranerupturecausedby aphasetransitionofthemembraneandchangeinitsfluidity(Anantaetal.,2005;Georgetetal.,2015).Thepressurelevelneededto achievea5log reductionofpathogenicmicroorganismindifferentfood-productsrangesfrom300to800MPa(Hendrickxetal., 10 2001)andoftensynergybetweenpressureandtemperatureisobserved(BuckowandHeinz,2008).Byincreasingtheprocesspres- sure,itispossibletodecreasethetemperatureneededtoachievethesameinactivation.Accordingtotheliterature,thepressure inducedeffectsleadingtocelldeathofvegetativemicroorganismscanbeattributedtofourfactors: (i) Proteinandenzymeunfolding,includingpartialorcompletedenaturation (ii) Cellmembranesundergoingaphasetransitionandchangeoffluidity (iii) Disintegrationofribosomesintheirsubunits (iv) Intracellular pH changes related to the inactivation of enzymes and membrane damage (Knorr et al., 2011a,b; Molina- Höppneretal.,2013;Georgetetal.,2015) Further,theinfluenceonthefoodmatrixstillneedsclarificationintermsofwhatcompoundsareformedordestroyedincompar- isontotheconventionalprocess.Also,aclearassessmentofwhatthepotentialandlimitsofHPPare,isstillmissingtothispoint. Somewell-knownpotentialandlimitsare: (i) AdvantagesandPotentials Rapid, quasi-instantaneous uniform distribution throughout the sample; Minimal or reduced thermal exposure; Instant temperature increase and subsequent cooling upon depressurization; Suitable forhigh moisture–content foods; in package processing; Suitable for both liquid and pumpable foods; Independent of product shape and size; Opportunity for novel productformulation;Distinctproductsthroughpressureeffectssuchasproteindenaturation,carbohydrategelatinization,and fatcrystallization;Withinsomepressure-thermalboundaryconditions,pressureacceleratesmicrobialinactivation;Consumer acceptanceasaphysicalprocess. (ii) Limits Batch or semi-continuous operation; Preheating step for pressure-assisted thermal processing (PATP) required; Thermal nonuniformity during PATP; Not suitable for products containing dissimilar compressibility materials such as foams; Throughputlimitedduetobatchoperation;Variableefficacyinenzymeinactivation;pressurealonecannotinactivatebacterial spores;Higherprocessingcosts. SomeauthorshavelookedbeyondtheobviousapplicationofHPP,otherthanjustmimickingofthermalprocessing.Anoverview ofsomeoftheapplicationscanbefoundintheliterature(Smelt,1998;Oeyetal.,2008;Verbeystetal.,2010;VanDerPlancken etal.,2012).Mostoftheseauthorsarereferringtothepromisinguseofhigh-pressureprocessingasagentlepreservationtechnique butarealsomentioningthepossibleapplications,ifthefoodsafetyisgiven,toaltertexture,increasethebioavailabilityofcertain healthpromotingcompounds,allergenicityreductionaswellasvitaminretention. Initially,theempiricapproachoftrialanerrorwasused,whichdrovetheevolutionofhighpressureprocessingfrominactivation studiesofvegetativemicroorganism(Hite,1899;TimsonandShort,1965;Saleetal.,1970;Metricketal.,1989;Cheftel,1995;Fior- ettoetal.,2005;Georgetetal.,2015)toinactivationofenzymes(Silaetal.,2008;Rauhetal.,2009;Knorretal.,2011b;Grauwet etal.,2012)toshuckingofcrustaceans(Kingsley2014)tosporeinactivation(Saleetal.,1970;Reddyetal.,2003;Blacketal.,2007; Olivieretal.,2012;SetlowandMarkland,2012;Reinekeetal.,2013a,b;Olivieretal.,2015;SevenichandMathys,2018)tostruc- ture/textureinducedbyhighpressure(Oeyetal.,2008;Silaetal.,2008;SikesandWarner,2016;Balasubramaniametal.,2016;Thai UnionGroup,2017;Simetal.,2019)tocurrentlythelatestfrontieroftheinfluenceonchemicalreactionspathways(e.g.,degra- dationofvitamins,oxidationofpolyunsaturatedfattyacidsandmitigationoffoodprocesscontaminants)(Oeyetal.,2008;Ver- beystetal.,2010;VanDerPlanckenetal.,2012;Sevenichetal.,2013).Toseebeyondtheobvious,onemustaskwhatisdifferent comparedtothethermalprocessesandwhy.Howcanthisdifferencebeusedasapotentialandadvantagetocreateforexamplenew functionalfoodingredients,foodpropertiesandhealthfoods?Someoftheseaspectsaretackledandcoveredbysomerecentreviews (Balasubramaniametal.,2016;Huangetal.,2017). Anotherissuethatneedsfurtherresearchfocuscouldbetothinkofnovelcombinationprocessesasforexampledoneinthepast withHPP/PEF,highpressurethermalsterilization,pressureassistedthermalsterilization(PATS),pressureinducedfreezing(PIF), pressureassistedfreezing(PAF),highpressurehomogenization(HPH)andhighpressureextraction(Volkertetal.,2008;Parketal., 2013;Huangetal.,2013;Sevenichetal.,2015a,b;SevenichandMathys,2018). Thisarticlewillreviewtherecentliteratureandwilldiveintodifferentareaswheretheauthorsconsiderhighpressureprocessing tohaveasignificantimpactinthecomingyears. 1.01.2 Vegetative Microorganisms, Spores, Viruses, Parasites, Bacteriophages and Nematodes 1.01.2.1 Vegetative Microorganisms TheuseofHPPtoinactivatepathogenicvegetativemicroorganismshasbeenlargelyinvestigatedforthepasteurizationofcommer- cialproductsfordecades(BuckowandHeinz,2008).In1899,Hite(1899)wasfirsttoconductexperimentsusinghighpressurein combinationswithfoodstoextendshelflife,andreportedthatmilkstayedsweetlongerafterthetreatmentwithhighpressure.Since OverviewofResearch Needs,Future andPotential ApplicationsofHigh-Pressure Processing 3 then,significantresearcheffortshavefocusedonunderstandingtheunderlyingmechanismsoftheinactivationofmicroorganisms underhighpressureconditions.HPPoffersalowerthermalinputintotheproductbycomparisonwithconventionalthermaltreat- mentandthereforeincreasesthequalityofthefoodwhilemaintainingfoodsafety(Smelt,1998;HoganandKelly,2005;Balasu- bramaniam et al., 2008; Bermudez-Aguirre and Barbosa-Canovas, 2011; Barba et al., 2012). Despite the steadily increasing commercial production of high pressure pasteurized food with several millions of tons per year (Aganovic et al., 2017), some importantscientificandtechnologicalquestionsarestillunsolved. Oneoftheseissuesistheimpactofdifferentintrinsicandextrinsicfactorsontheinactivationmechanismsofvegetativebacteria andbacterialsporesunderpressure.Tounraveltheimpactofthedifferentpressureandtemperaturecombinationsonapossiblecell deathorrecovery,detailedanalysesaboutthephysiologicalstateofthecellsandhowtheyareinfluencedbydifferentfoodcompo- nents are needed, In recent years, a common world-wide concern has been foodborne outbreaks with pathogens like EHEC (O157:H5;O104:H4)andothers(AdleyandRyan,2016).Thecontaminationofthefood supplywith spoilageandpathogenic microorganismscontinuestobeaglobalproblem,despitethewiderangeofpreservationmethodsemployed(Kaczmareketal., 2019).Despitesignificantadvancesinfoodprocessingtechnologies(hurdleconcept,newinnovativenon-thermaltechnologies), anannualestimated76millioncasesoffoodborneillnessesoccurintheUSalone,resultinginapproximately5000deaths(Scallan etal.,2011).ThereportbytheUSCenterfordiseasecontrolandpreventioncenterstatesthatmost(58%)illnesseswerecausedby norovirus,followedbynontyphoidalSalmonellaspp.(11%),Clostridiumperfringens(10%),andCampylobacterspp.(9%).Leading causesofhospitalizationwerenontyphoidalS.spp.(35%),norovirus(26%),C.spp.(15%),andToxoplasmagondii(8%).Leading causesofdeathwerenontyphoidalS.spp.(28%),T.gondii(24%),Listeriamonocytogenes(19%),andnorovirus(11%).Intheyear 2000,e.g.,approximately2.4millionpoundsofbeefwererecalledduetopossiblecontaminationwithEscherichiacoliO157:H7 (Kaczmarek et al., 2019). Newest studies from the WHO (2015) on foodborne diseases caused by pathogenic microorganisms, suchasSalmonella,Campylobacter,EHECandNorovirus,showthatworldwide1in10peoplefallilleveryyearfromeatingcontam- inatedfoodand420,000dieasaresult.Here,theapplicationofhighpressureincombinationwithmildtemperaturesatpressures between300and700MPacouldpossiblybeusedtoinactivatevegetativepathogenicmicroorganismsaswellasseveralenzymes, whichcausefooddeterioration.TheresistancetowardtemperatureofthepathogenicEscherichiacolistrainsisusuallyhigherthan itsnon-pathogeniccounterpart(Garcia-Graellsetal.,1998).Hence,amoreintenseheattreatmentneedstobeapplied.Themore complexandresistantthemicroorganismthemoreintensemustbethetreatment.IncomparisontothepathogenicE.coli,higher temperaturesandhigherpressuresmustbeappliedtoinactivatespores(Reinekeetal.,2013a,b).Thisbecomesevenmorecomplex if this is conducted in a real food system since here baro-protective effects can occur and the severe heat treatment can lead to unwantedchangesinthefoodmatrix,leadingtotheformationofunwantedandpossiblyunhealthycompoundsinthefoodstuff. Thecomparisonofpressureresistanceamongvegetativefood-bornepathogensrevealedthatstrainsofEscherichiacoliO157:H7were themostresistantsofarencountered.TheUSDA(2012)hasrequirementsofE.coliO157:H7astheindicatorstrainforreprocessing, anHPPprocessthatachievesa5-log E.coliO157:H7reductionshouldbesufficientenoughtoproducemicrobiallysafeproducts. 10 OtherpossibleindicatorstomonitorifHPPissufficientlyappliedduringprocessingisbycoppertabletsincombinationwith Heckelvalue(rateofdensitychangeofe.g.,chopperunderpressure).Theincreaseindensityovertheentirepressurerangeislinear. Thetabletscanbeplacedinsidethevesselaswellasdirectlyintothefood.Measurementsat400MParevealedthatfortrialswith hamtheproductexperienced9MPalessthanthesurroundingwater.Thiscouldleadtoshortcomingsintermsofsufficientinac- tivation.Thetrialwasnotrepeatedat600MPa(MinerichandLabuza,2003). Anotherphenomenonthatcouldleadtofalseresultsintermsofinactivationkineticisagglomeration,whichisverycommonfor vegetativemicroorganisms aswellasspores.Theeffectivenessofinactivation processesisoftenverifiedbychallengetestsusing foodsthathavebeenspikedwithhighlyconcentratedmicrobesuspensions. Due topreparation, storageandhandling ofthose suspensions,theclumpingandtheformationofaggregatescanhardlybeavoided.Thisphenomenoniswellknown,firstreported ontheeffectofsporeagglomerationsoninactivation.However,theimportanceforthequantitativeassessmentofsurvivorsininac- tivationexperimentshasrarelybeenaddressed.Itsimportancebecomesevidentbythefollowing:Agglomeratesproduceonecolony forahighnumberofcellsupto103CFU’s.Consequently,agglomeratesofunknowncellnumbersarealwayscountedasonespore untilallsporesintheagglomerateareinactivated.Beyondthis,agglomerationanddisintegrationcanchangethecolonyforming unitspermilliliter.Inthiscontextamodelforthediscussedphenomenonwasdeveloped(MathysandHeinz,2006). 1.01.2.2 Spores Theinactivationofbacterialendosporesbypressureisgenerallyconsideredtorelyonpressure-inducedsporegermination,followed by inactivation of germinated spores (Setlow, 2003; Margosch et al., 2004; Margosch et al., 2004). In the past decades, other possiblenon-physiologicalpathwaysofsporegerminationhavebeendetected.Non-nutrientgerminationcanbefurthercatego- rized into a (recently discovered) second physiological and several non-physiological routes. The physiological routes include germinationinitiatedbyeukaryotic-likeserine/threoninekinase,whichislocatedintheinnersporemembranelikenutrientrecep- tors.Thiskinase,whichispresentinBacillusandClostridiumspecies,recognizespeptidoglycanfragments.Non-physiologicalgermi- nationpathwaysinitiatesporegerminationbybypassingindividualgerminationsteps.Thiscouldbestimulatedbyphysiochemical agents,suchasexogenousCa-DPA(Paidhungatetal.,2002;Moir,2006),whichdirectlyactivatestheCLECwlJ,orcationicsurfac- tantssuchasdodecylamine(Setlow,2003),whichinterfereswiththeinnersporemembraneandcausesadirectreleaseofCa-DPA. Atpressuresbetween100and400MPaithasbeenshownthatthenGeRofBacillussubtilisandBacilluscereusaretriggered.Thespores germinatedquitewellbetweenpressuresof100–200MPaandledtoamaximum4log inactivationbutthepressureinduced 10 4 Overview ofResearch Needs,Future andPotential ApplicationsofHigh-Pressure Processing physiologicalgerminationdecreasedforhigherpressures,indicatingthatotherpathwaysmustbeactive(Wuytacketal.,1998;Paid- hungatetal.,2002;Reinekeetal.,2013a,b).Atreatmentof200–400MPaat40(cid:1)Cfor30minshowedgerminationofthesporesbut negligibleinactivation(Wuytacketal.,1998). Toachieveaquickandsuddeninactivationofspores,whichisrelevantforfoodprocessing,pressuresabove500MPamustbe appliedincombinationwithtemperaturesabove60(cid:1)C.Undertheseconditions,Paidhungatetal.(2002)wereabletogerminate B.subtilissporesthatlackallmajornutrientreceptors.Thissuggestsadirectopeningofthespores’Ca–DPAchannels,agermination mechanismidentifiedasactiveat200MPaandmoderatetemperatures(<50(cid:1)C)usingB.subtilismutantstrainsthatlackthenGeR. ThesefindingswerealsoverifiedbyReinekeetal.(2012)forpressures(cid:3)600MPaandtemperatures(cid:3)60(cid:1)C.Thefollowingstep, whichisrapidreleaseofCa–DPAunderpressure,isaccompaniedbycorehydration.Thisstepofgerminationisthecrucialstepwith regardtolossofresistance,anditisthereforeofgreatinterestforavarietyofsterilizationtechniquesandresearch(Reinekeetal., 2013a,b).Therefore,theabilityofthesporetoretaintheDPAaslongaspossibleundertheseconditionsbecomestheratelimiting stepofthesporeinactivation(Margoschetal.,2006;Reinekeetal.,2013a,b).ThissuggeststhatthestructuremostsusceptibletoHP (600MPa) and high temperatures (60(cid:1)C) is the inner spore membrane or its associated membrane proteins (Reineke et al., 2013a,b). At pressures above or equal to 400MPa, when an opening of the Ca - Dipicolinic acid (DPA) - channels occur, the followinghappens:(i)DPAisreleasedfromthesporecore;(ii)thesporecoregetshydrated;and(iii)thesporebecomesthermo- andpressuresensitiveandcanbeinactivated(Reinekeetal.,2012).Further,athresholdpressureof600MPawasestablished;atand abovethispressureleveltheDPA-releaseisdominatedbytemperature.Toguaranteeasuccessfulinactivationofsporesbypressures of600MPa,manyresearchersrecommendatreatmentat90–121(cid:1)C(Margoschetal.,2004;JulianoandBarbosa-Canovas,2005; Margoschetal.,2006;Mathysetal.,2007;Knorretal.,2011b;Georgetetal.,2015;Sevenichetal.,2016)duetothesynergisticeffect pressureandtemperaturehaveonthesporeinactivation(Olivieretal.,2015).Incomparisontoconventionalretorting,thiscould reducethethermalloadappliedtotheproductwithoutaffectingthesafetyorthequalityofthefood.Twosterilizationapproaches canbederivedfromthis,bothofwhichneedtheadiabaticheatofcompressiontoreachthetargettemperature: (cid:129) Pressureassistedthermalsterilization(PATS):pressureisneglectedandonlyseenasthemethodtoreachtheendtemperature faster(Dunneetal.,2010) (cid:129) HighPressureThermalSterilization(HPTS)whichtakesintoaccounttheimpactofpressureonthesporeinactivation(Mathys, 2008;Knorretal.,2011) Recently,thedifferencesingerminationunderpressurebetweenClostridiumsporesandBacilluswererevealed,showinginteresting insights(Paredes-Sabja et al., 2011; Lenz and Vogel, 2015; Doona et al., 2016). InBacillus spp.,DPArelease triggers cortex lytic enzyme (CLE) activation; CLE action is not essential for DPA release, but it can accelerate it. In Clostridium difficile or C.perfringens,theinitiationofcortexhydrolysisbySleCprecedesandtriggersDPArelease.UsingC.difficilespores,whichlackinner sporegerminantreceptors,550MPatreatmenttriggeredDPArelease.However,sporegerminationwasnotcompletedbecausecortex hydrolysis is notactivated by DPA release. C. perfringens spores became activated by high pressures during thecome-up time to 550MPa.Itcouldbeshownthat(i)theactionof550MPadirectlystimulatedDPAreleaseand(ii)activationofinnermembrane germinantreceptorsduringthecome-uptimeto550MPaandthatthisactivatedstateofgerminantreceptorsismaintainedforawhile andactivatescortexlyticenzymes(Doonaetal.,2016).Thisisonlyvalidiftemperaturesbelow80(cid:1)Careused;ifaheatshockT >80(cid:1)Cisapplied,sporegerminationcanbecompleted.Thesefindingswereveryinterestingsincetheyshowedhowdifferentspores areandhowimportantprocessknowledgeandmicrobiologicalknowledgearetocreatesafeprocesses,suchastheHPTS. Thequestionofwhatkindofindicatorstrainshouldbeusedistothispointstillnotsettled.Someresearchersusethenon- pathogenicBacillusamyloliquefaciens,insteadofClostridiumbotulinum,asasurrogatetocheckforsufficientinactivationrespectively sterilizationsuccess(Olivieretal.,2015;Margoschetal.,2006;Sevenichetal.,2016).Sinceotherstrainsusuallyusedtotestfor sterilizationlikeGeobacillusstearothermophilusandClostridiumsporogenesareveryhighpressurehightemperaturesensitive(Sevenich etal.,2016).Itisalsoimportanttonotethatonecannotconcludebasedoninactivationkineticsobtainedinbufferormodelfood systemshowtheinactivationofsporeswillbeinrealfoodsystems(Georgetetal.,2015;Sevenichetal.,2015a,b;Sevenichand Mathys,2018).TheUSFDArecommendstestingfora4log inactivationofClostridiumbotulinum(Dunne,2009;Balasubrama- 10 niamandLelieveld,2016). 1.01.2.3 Viruses, Bacteriophages, Parasites andNematodes Intotal,thereare31knownpathogensthatcausefoodbornediseases,21arebacteriarelated(E.coli,Salmonella,Listeriaetc.),five virusesandfiveparasites(AdleyandRyan,2016).Especiallyvirusesandparasitesaregenerallyunderrecognizedifitcomestofood bornediseases.Thegrowingglobalinterconnectionofthefoodsupplychainandmoresophisticatedanalyticaltoolsarereasonsfor theincreaseddiagnosisoffoodbornediseases(Dornyetal.,2009).Foodbornediseasesalsohaveanimpactontheeconomy,each incidentwascalculatedwith(cid:4)1500$/personandatotalannuallyestimatedcostof$75billion,intheUnitedStatesin2015alone (Moyeetal.,2018).Beforegoingintofurtherdetailonhowhigh-pressureprocessingmighthaveanimpactontheinactivationof theseorganisms,ashortintroductiononwhatdefinesthesegroups,wheretheycancomeincontactwithfoodsandwhatdisease theycancause,isgiven. (i) Viruses/Bacteriophages:Avirusisasmallinfectiousvehiclethatcannotgroworreproducewithoutalivingcell.Avirusinvades livingcellsandusestheirchemicalmachinerytokeepitselfaliveandtoreplicateitself.Itmayreproducewithfidelityorwith OverviewofResearch Needs,Future andPotential ApplicationsofHigh-Pressure Processing 5 errors(mutations);thisabilitytomutateisresponsiblefortheabilityofsomevirusestochangeslightlyineachinfectedperson, makingtreatmentdifficult.Incommonusage,thetermvirusisusedtodescribevirusesthatcanonlyaffecthumans.Viruses causemanycommonhumaninfectionsandarealsoresponsibleforseveralrarediseases.Thetwomainfoodbornevirusesof concerninthefoodindustryaretheNorovirusandHepatitisAvirus.In2011,theNoroviruswasthemaincauseoffoodborne illnessesintheUnitedStates(AdleyandRyan,2016;Panetal.,2016). Therearemainlytwofoodtypesthathaveahighriskforcontaminationduringproductionorharvest.Onewouldbefruitsand vegetables,wherethecontaminationcanbeduetotheuseofnon-potablewaterforcleaningorfecallycontaminatedfingers (Kingsley,2013).Thesecondfoodtypehavingelevatedriskofcontaininghighamountsofvirusesareshellfish(oysters,clams, cocklesetc.).Duetotheirnatureasfilterfeedersandthehighamountsofvirusesfoundinseawater(9(cid:5)108virons/mL),they can accumulate viruses as much as 1000-fold (Kingsley 2013, 2014). Bacteriophages are viruses that can only attack and replicateinbacteriaandarchaea.Phageshavebeenusedsincethelate19thcenturyasanalternativetoantibioticsintheformer SovietUnionandCentralEurope,aswellasinFrance.Theyareseenasapossibletherapyagainstmulti-drug-resistantstrainsof many bacteria (phage therapy). Since 2006, the United States Food and Drug Administration (FDA) and United States Department of Agriculture (USDA) have approved several bacteriophage products. LMP-102 (Intralytix) was approved for treatingready-to-eat(RTE)poultryandmeatproducts.Inthatsameyear,theFDAapprovedLISTEX(developedandproduced byMicreos)usingbacteriophagesoncheesetokillListeriamonocytogenesbacteria,inordertogivethemgenerallyrecognizedas safe(GRAS)status.InJuly2007,thesamebacteriophagewasapprovedforuseonallfoodproducts.In2011,USDAconfirmed thatLISTEXisacleanlabelprocessingaid.Researchinthefieldoffoodsafetyiscontinuingtoseeiflyticphagesareaviable optiontocontrolotherfood-bornepathogensinvariousfoodproducts(Moyeetal.,2018). (ii) Parasites including nematodes: Parasitism is a relationship (consumer-resource interaction) between species, where one organismlivesonorinanotherorganismontheexpenseofthehost.Parasitesincludeprotozoans,tapeworms,nematodesetc. Someofthemajorparasitesthatareofconcernforthefoodindustryaree.g.,Echinococcusmultilocularisassociatedwithberries andwater;Cryptosporidiumspp.arebeginningtobeassociatednotonlywithwater,butalsowithsalads;Trypanosomacruziis foundinjuices;Trichinellaspp.arenowadaysfoundingameanimals;Anisakiasisisbecomingaglobalproblemastheworld developsatasteforsushi(Robertson,2018). Literatureonthepressureinactivationofvirusesisscarceandthereareonlyahandfulofpapersthataredealingwithinactivation studiesofvirusesinfoods(Kovaetal.,2010;Kingsley2013,2014;Balasubramaniametal.,2015).Especiallyresearchdealingwith theinactivationmechanismarelimited(Panetal.,2016).Researchwithhumannorovirusstrainsandvirusesingeneralisdifficult duetothelackofsuitablelaboratory animalsandtheinabilitytopropagatethevirusinvitro.AstudyconductedbyWiezorek (2012)withPixunavirus,whichisasurrogateformanyhumanparthenogenicalphavirus,showedthatPixunacouldbeinactivated by109withatreatmentat150MPa/37(cid:1)C. Theinactivationofvirusesviahighpressureprocessingisassociatedwithalteringtheproteinslocatedatthecapsidofthevirus. DuetoconformationchangeoftheseproteinsthevirusisnotabletodockontothehostcellandreleasetheRNAintothecell (Moronietal.,2002;Kovaetal.,2010;Kingsley,2013;Louetal.,2015).Non-envelopedvirusesaregenerallymoresensitivetoward highpressureprocessingincomparisontoenvelopedviruses,whichhavealipidenvelope.Theinactivationofvirusesisdepending, asvalidforsporesandbacteria,onthetemperature,pressureandtimeapplied(Panetal.,2016).Pressuresabove400MPaareeffi- cientenoughtoinactivatemostviruses(Kovaetal.,2010).Although,higherpressuresdonotnecessarilyneedtoleadtoamore efficientinactivationofamoreresistantvirus. Thestudiesfoundintheliteraturesuggestthatviruseslikehumannorovirus(Norwalk8fIIb)inoculatedinoysters(104)canbe completelyinactivatedby600MPaand5min(Leonetal.,2011).StudieswithHepatitisAvirusalsoinoculatedinoysterswith106 showedaninactivationofmorethan3log after400MPaand1min(Kingsley,2013).Rotavirusinbuffersolutionwasinactivated 10 by8log at300MPaand2min(KhadreandYousef,2002).VerypressureresistantarethevirusesAichivirusandPoliovirus,which 10 werecompletelyresistantat600MPaand5min(Kingsleyetal.,2002). Forviruses,theUSFDAisproposingamaximumdesiredreductioninviruslevelof5log (Panetal.,2016). 10 Theliteratureonnematodesandotherparasitesisveryscarceandvirtuallynon-existing.Thereisonepublicationontheinac- tivationofapinewoodnematodeinwoodchips,inoculatedwith1.2(cid:5)105byhighpressureprocessing.Hereitwasshownthatthe nematodeswerecompletelyinactivatedby30MPaataholdingtimeof5min(Fonsecaetal.,2014).Thissuggeststhatthebiggerthe organismtheeasieritcanbeinactivatedbyhighpressureprocessing.Thequestionthatstillneedstobeansweredisifthatisalso validforotherparasiteslikeCryptosporidium,Trypanosomaetc. Theresearchontheinfluenceofhighpressureprocessingonvirusesandparasitesneedstobedrivenforwardsinceitisaprom- isingalternativetoguaranteethesafetyofusuallynon-processedorgentleprocessedfreshproducts. 1.01.3 Chemical Reactions: Influence on Allergens, Toxins (Food Borne and Agriculturally Based) 1.01.3.1 Allergens Allergyisafalse,hypersensitiveandintenseimmuneresponsetowardtypicallyharmlesssubstancesintheenvironment.Foodcan beatriggerfortheimmunesystemtooverreact,especiallydairy,egg,soy,peach,cherry,selfish,wheat,nutsandapplecancausean allergicreaction(SomkutiandSmeller,2013).Foodallergyismoreprevalentinyoungchildren(5%)but3%–4%ofadultsalso exhibitaformoffoodallergy. 6 Overview ofResearch Needs,Future andPotential ApplicationsofHigh-Pressure Processing ThebeginningofatypeIallergicreactionisthesensitizationtoanallergen.Theinitialcontactofanallergenwiththemucosaof asusceptiveorganismisfollowedbyacomplexseriesofevents,leadingtotheproductionofallergenspecificimmunoglobulinE (IgE).Theeffectorphaseofanallergicreactionisinitiateduponsecondexposuretotheallergen.AllergenbindingtothespecificIgE antibodyonthemastcellswillcauseaninflammatoryresponseduetothereleaseofhistamineandothermediators.Notallaller- gensareabletosensitize,i.e.,inducetheproductionofspecificIgE-typeantibodies(SomkutiandSmeller,2013;Ontiverosetal., 2015;Ekezieetal.,2018).Allergenswhicharecapabletobothsensitizeandtriggerallergicreactionsarecalledcompleteallergens. CrossreactiveallergensbindtoIgEantibodiesthatarepresentinthebodyduetoanearliersensitizationbyanotherallergen.There aremorethan6000allergenswhichcanhaveanegativeimpactandtherearecurrentlynoacceptedtherapeuticapproachesother thanavoidingthefoodsthatcontainthepossibleallergen(Ontiverosetal.,2015;Meinlschmidtetal.,2016).Here,thetargeted treatmentbyfoodprocessingtechnologies,suchashighpressure,enzymatichydrolyzes,heattreatmentorcombinedprocesses, couldbeapromisingstrategytocreatehypoallergenicfoods.Togainknowledgeifafoodproteinisallergenicornotitneedsto becharacterizedbytheallergic(epitopes)andnon-allergiccomponents;thiscanbedonewiththehelpofimmune-informatics (Ontiverosetal.,2015).Themainsequence/patterninallergenicproteins’secondarystructureare(1)antiparallelb-strands;(2) antiparallelb-sheets;(3)aþbstructuresand(4)a-helicalproteins(SomkutiandSmeller,2013).Sincehighpressureprocessing can have an impact on the functionality of proteins by altering the secondary, tertiary and quaternary structure of the protein duetochangesinelectrostaticandhydrophobicinteractionsitsurelyinfluencesthereactivepropertiesofallergenicproteins(Jimé- nez-Saizetal.,2015).Theapplication,alsoincombinationwithotherprocesses,isaninterestingtooltocreatefoodswithreduced foodallergenicity.SomkuttiandSmeller(2013)mentionedintheirreviewthatthreequestionsneedtobediscussedtofindoutif highpressureissuitable:(1)Howhighisthepressureneededtounfoldtheprotein?(2)Istheunfoldingreversible?and(3)Howis thebindingofIgEtotheallergen? Ingeneral,itcanbesaidthatforsomeofthemajorfoodallergenslikeMalD1(apple),Bosd5(Milk),Gald2(Egg),Arah2 (Peanut),Gadm1(fish),pressuresandtemperaturesof150–600MPaand30–80(cid:1)Careneeded(SomkutiandSmeller,2013).The mechanismofapotentialallergenicreductionwhileapplyinghighpressureprocessingiseitherthemaskingofepitopesdueto conformationalchangesinducedbyhighpressure,orthehigherexposureofepitopesbutthereforeleadingtohigherenzymatic hydrolyzes(Peñasetal.,2008;Meinlschmidtetal.,2016).Thesecondmechanismwillonlyworkifthefood/allergensaretreated withenzymeslikepepsin,chymotrypsinorpapainafterthetreatment,otherwiseitwouldmakethefoodevenmoreallergenic.It could be shown that the allergenic potential of the walnut allergens Jug r 1–5 can be reduced by pressures ranging from 550– 650MPa(Ekezieetal.,2018).Furtheradecreaseof40%–60%ofalpha-caseininmilkandPrup3inpeachcouldbeshowninapres- surerangeof200–600MPa(Lavillaetal.,2016;Ekezieetal.,2018). Highpressurealonecouldnotmodifytheallergenicpropertiesofcarrots,peanut,milk,celery,apple(Jiménez-Saizetal.,2015). Asitissuitableforothertechnologies,themosteffectivewaytoreducetheallergenicitybyhighpressureprocessingisthecombi- nationwithotherprocessesthatactsynergistically.Onepossibilityistocombinepressureandheat,whichworkswell,butmore effective seems to be thecombined treatment of high pressure and enzymic hydrolyzes during or after the treatment (Somkuti andSmeller,2013;Jiménez-Saizetal.,2015;Meinlschmidtetal.,2016).Meinlschmidt(2016)studiedtheeffectonsoyprotein isolate with an enzymatic hydrolysis during and after high pressure processing at pressures ranging from 100 to 600MPa at 50(cid:1)Candadwelltimeof15min.Itwasshownherethatacombinedprocessatpressuresfrom300to600MPacouldleadto anearlycompletelossofimmunoreactivityofthementionedallergen.Further,theoilbindingandfoamingactivitywereenhanced. SimilarresultsandstudiesaresummarizedbySomkuttiandSmeller(2013),e.g.,beta-lactoglobulinandovalbuminincombina- tionwithpepsinledtoanincreasedhydrolysisat600MPaandafewminutesoftreatmenttime.ForpeanutallergenArah6no changescouldbeshownat700MPaat20or80(cid:1)C. Thepressurestabilityofallergenproteinsvariesgreatly,dependentontheirsecondarystructure,thesurroundingsandtreatment conditionsapplied.Further,combinedstudiesareneededtoassesstheinfluenceandtheeffectivenessofpressure-enzymatichydro- lyzesprocesses.Eventually,onlyprickteststudiesincombinationwithstructuralanalysisoftheallergenwillvalidatetheeffective- nessofthereduction. 1.01.3.2 Toxins Therearetwotypesoftoxinsthatareofinterestinfood.Onebeingthoseincorporatedduetotheuseofchemicalsduringgrowthof crops(pesticidesandherbicides)aswellasthegenerationofmicrobiologicaltoxins,suchasaflatoxins,C.botulinumtoxin,etc.The secondonebeingtoxinsthatariseduringtheprocessingofthefoodsduetoheattreatment,theso-calledfoodprocessingcontam- inants(furan,acrylamide,HMF,MCPD-estersetc.) 1.01.3.2.1 Food Processing Contaminants TheoccurrenceofFPCsinfoodsisnotnew,astheyhavealwaysexistedsincethefirstdayhumansstartedtoprepareandconserve theirfoodsbyfireorheat.Nowadays,theawarenessofwhatthesecompoundscandowithinthehumanbodyismoreadvancedand theanalyticaltoolsareavailabletoanalyzetheamountsofthesecompoundswithinfoodsinthemgkg(cid:6)1range(Sevenich,2016; Sevenichetal.,2016). Thehightemperaturesat>110(cid:1)Cneededduringthermalsterilizationfortheinactivationofsporesleadtotheformationof unhealthycompoundswithinthefood.Sincethebeginningofthenewmillennium,moreattentionhasbeengiventothemech- anisms and mitigation strategies of these compounds. The way of our foods from farm to fork or from raw material to better OverviewofResearch Needs,Future andPotential ApplicationsofHigh-Pressure Processing 7 digestiblesafefoodsismadepossiblebyprocessessuchasheattreatment.Thosetreatmentsoftenresultinanover-processingand, duetochemicalchangesintheproduct,givingrisetothefoodprocessingcontaminants(FPCs);synonymsare,process-induced toxicants and neo-formed contaminants. FPCs only involve those compounds formed during the processing (heating, roasting, frying,baking,grillingetc.)ofthefood.FPCsaresubstancespresentinfoodbecauseoffoodprocessing/preparationthatareconsid- eredtoexertadversephysiological(toxicological)effectsinhumans,i.e.,substanceswhichposeapotentialorrealrisktohuman health.Ingredientscommonlyoccurringinfoodformulationsareexcellentsubstratesforchemicalreactionsoccurringunderthe conditionsencounteredinfoodprocessing.Thereactionproductsformeddependontheprocessesandconditionsused,suchas fermentation,irradiation,orheatprocessing(StadlerandLineback,2009).Thecompoundsformedplayavitalroleinfoodprop- ertiessuchasflavor,aromaandcolor.PrecursorsofFPCsaresugarsandaminoacids;otherreactionpathwayscaninvolvepoly- unsaturated fatty acids (PUFAs; linoleic acid), ascorbic acid, sugars (glucose, fructose), glycerol, chloride or carotenoids (Crews and Castle, 2007; Vranová and Ciesarová, 2009; Lachenmeier and Kuballa, 2010; Bravo et al., 2012; Crews, 2012). Therefore, changingtherecipeoffoodsisnotanoptionsincesomeofthemostpotentprecursorsofFPCsareessentialnutrients,likepoly- unsaturated fatty acids, reducing sugars, carotenoids, proteins etc. Compounds formed during the processing of food are, for example, acrylamide, furan, 3-MCPD esters etc. These show carcinogenic, mutagenic (genotoxic), or neurotoxic properties at highdosesinanimalstudies(BfR,2004,2012;Studeretal.,2004;Märketal.,2006;VranováandCiesarová,2009;Jestoietal., 2009;Larsen,2009;StadlerandLineback,2009;LachenmeierandKuballa,2010;Palmersetal.,2014;Sevenichetal.,2015a,b;Ket- tlitzetal.,2019).Therefore,ariskforhumans,especiallyforinfants,theelderlyandimmunesuppressedpersonisnotneglectable. TheriskoftheexposuretoFPCsisnotanewone,sincehumansalwayshavebeenexposedtothesekindsofcompoundsfromthe moment“theycaughtfire”.Nevertheless,thereisapublicconcernandthoserisksmustbeminimized(Curtisetal.,2013).Forgen- otoxicandcarcinogenicsubstances,suchasfuranandacrylamide,theALARAprinciple(aslowasreasonablyachievable)isapplied tofoodsasapossibleriskassessment(CrewsandCastle,2007).Nevertheless,thedatashownhereclearlyindicatesthattheFPCsof majorconcerninfoodsareacrylamide,furanand3-MCPD.TheEuropeanCommissionandtheEuropeanFoodSafetyAuthority (EFSA) have been monitoring different FPCs, especially acrylamide, furan (and its derivates), 3-MCPD and -esters and glycidyl esters,inallkindsoffoodsoverthelastyearsandhavealreadyissuedbenchmarklevelsforacrylamidein2017(EU,2017).The creationandintroductionofnewguidelinesinthefutureislikelyandwillbeadifficulttaskforthefoodindustry(EFSA,2013; Kettlitz et al., 2019). Therefore, other technologies and research is needed to find mitigation strategies which lead to the same quality without affecting the safety of the product. Here the high-pressure thermal sterilization or high-pressure pasteurization (insteadofconventionalheattreatment),ohmicheatingandvacuumbaking,justtonameafewinnovativetechnologies,could bepowerfultoolstoachievethisaim.Researchinalltheseareasisprogressingatarapidpaceandtheseselectedexamplesshow thatprocesstoxicantshaveinthepastfewyearsgainedsignificantattentiononaglobalscaleintermsofpotentialhumanhealth risk.InMarch2018,EFSAissuedacallforthecontinuouscollectionofchemicalcontaminantsoccurrencedatainfoodandfeed (EFSA,2018).ThesedataareusedinEFSA’sscientificopinionsandreportsoncontaminantsinfoodandfeed.Thislastcallshows howimportantitistolegislativeorganizationstobeontopofthissensitivematter.Thereareonlyafewstudiesavailablethatshow themitigatinginfluenceofhigh-pressurethermalsterilizationonthereductioninrealfoodsystems.Sevenichetal.(2013)andSev- enichetal.(2015a,b)showedthatareductionoffuranindifferentfoodsystems(vegetablebabyfoodandsardineinoliveoil)is possiblewithHPTS(600MPa,90–121(cid:1)C)byupto95%incomparisontothermalsterilization.Palmersetal.(2014)alsoshowed areductionoffuraninvariousvegetableblendsunderhighpressurethermalsterilizationconditions(600MPa,117(cid:1)C)incompar- isontothermaltreatment.Tothispoint,thequestionifthepressure,followingtheLeChatelierprinciple,hasaninfluenceonthe formationpathwayofthesecompoundsisstillunanswered.IntheopinionoftheauthorsthereductionofFPCscanbesolelyattrib- utedtothelowerthermalloadappliedtotheproduct.Forhighpressurepasteurizationtheformationoffoodprocessingisnotan issuesincethethresholdtemperaturesofabove110(cid:1)Corhigherarenotreached. 1.01.3.2.2 Aflatoxins, Pesticides andHerbicides Tothisdatethereisnoliteratureavailablehowhigh-pressureprocessingaffectsordestroyseitheraflatoxinsorpesticidesandherbi- cides.Itwillbeinterestingtoinvestigatethesecompoundsandhowtheymaybealteredbyhighpressureprocessing.Aflatoxinfor exampleisverytemperaturestableandstartsdecomposingattemperaturesbetween237and306(cid:1)C(Kumaretal.,2017)butaheat treatmentbetween90and120(cid:1)Ccanalreadyreducetheamountofaflatoxinasmuchas25%–65%.Therearesomestudiesinthe publicdomainonprocessingbyextrusioncookingofpeanutandricemealattemperaturesof140–200(cid:1)Candpressuresinthe extruder of 30–60bar. The highest aflatoxin reduction was found to be 51%–95% with a moisture content of 35% in peanut meal, and the extrusion variables did not significantly affect its nutritional composition (Castells et al., 2006; Kumar et al., 2017).ItisworthwhiletoinvestigatetheeffectofHPTSandHPPonaflatoxins.Therewasnopublicationfoundoninfluenceof high-pressureprocessingonpesticidesorherbicides. 1.01.4 Process-Structure-Relationship Highpressureprocessingcanalterthepropertiesofmacromoleculeslikestarch,pectinandproteinsandcombinationsoftheafore- mentionedcompounds.Therefore,itcanbeofgreatinteresttocreatenewtextures,whichcouldleadtothedevelopmentofnewand excitingstructures.Thisisnotonlytodesignnewfoodsbutalsotoevaluatehowtheprocessinfluencesproductsintermsoftexture andstructurethatusuallywouldhavebeenthermallytreated.Thereissignificantinterestinunderstandingtheeffectsofhighpres- sureonfoodandfoodingredients,toanticipatefurtherapplicationsofthetechnology(ETP,2007;SunandHolley,2010).The followingsectionwillsummarizeongoingresearchactivitiesandneedsinthisfield. 8 Overview ofResearch Needs,Future andPotential ApplicationsofHigh-Pressure Processing 1.01.4.1 Starch Starchisoneofthemajorcarbohydratesusedinthefoodindustryandinhumandiet.Asasourceofenergyandamajorsourceof carbon,starchisanessentialpartofhumannutrition.Inthefoodindustry,starchservesasathickeningandgellingagent,e.g.,for saucesandpuddings.Starchanditsderivativesarealsoimportantinthechemical,textileandpaperindustries.Itisobtainedfrom thereserveorgansofplants.Wheat,potatoes,maizeandtapiocaarethemostimportantrawmaterialsinglobalstarchproduction. Asalreadymentioned,ultra-highpressurehasasignificantimpactonthephysicalandchemicalpropertiesofmacromolecules.In termsofcarbohydrates,ultra-highpressureleadstogelformation,aloweringofthegelationtemperature, areducedenzymatic stabilityofstarchandlittleornoMaillardreactionatroomtemperature(Hendrickxetal.,2001).Partialswellingandgelatinization ofstarchcanoccurunderhighpressureconditionsevenatroomtemperature(Stute,1999;Douzalsetal.,2001;Katopoetal.,2002). Therequiredtreatmentparameters,suchaspressure,temperature,holdingtimeandwaterconcentrationdependtoalargeextenton thetypeofstarch(Autio,1998;BauerandKnorr,2005).Thegelatinizationunderultra-highpressureatroomtemperatureisaccord- ing to Rubens et al. (1999) described similar to thermal gelatinization by a 2-step mechanism. First, the amorphous areas are hydrated, which loosens the crystalline structures and swells the grain. In the second step, the crystalline areas are increasingly brokenupandhydrated.Onlyasmallamountofamyloseleakagefromthegrainscanbeobserved.Stoltetal.(1997)alsodescribe stabilizationofthecrystallineareasthroughinteractionswiththeremainingamylose.HydrogenbondsandvanderWaalsforcesare likelystabilizedbyultra-highpressure,whichinturnfavorsthedoublehelix(Buckowetal.,2007).Thegranularcharacterofthe grainsislargelyretainedduringtheultra-highpressuretreatment(Hayashi,1992)andthereisnocompletedisintegrationofthe structureasinthecaseofthermalgelatinization.Thus,thepressure-induced,pastytosolidstarchgelscanonlybereferredtoas particlegels,whichdonotformarealgelnetwork.Inaddition,thethermallyformedstarchgelsandpastehaveamuchhigher strength or viscosity (Buckow et al., 2007). Interestingly, the starch grains lose the double refraction despite no degradation of thecrystallinestructures.Itbecomesclearthattheprocessdescribedrequiresanexcessofwatermoleculesandisthereforeheavily dependentonthewatercontent.Additionsofwater-bindingsubstancessuchassugarorsaltscanthereforehaveasignificantimpact onthehigh-pressure-inducedswelling.RumpoldandKnorr(2005)showedthatthepreviouslydescribedinfluenceofthenumber ofequatorialgroupsalsoexistsinthecaseofswellingandgelatinizationinducedbyhighpressure.Starcheswithahighamylose- amylopectinratio,suchaswheatorpotatoes,haveveryhighpressureresistance(Hendrickxetal.,2001).Waxystarchestherefore showlittleresistancetopressure(Simoninetal.,2009).Stute(1999)usedlossofbirefringencetoshowthatpressureresistanceis alsovisibleintheX-raydiffractionpattern.Accordingly,mostlyB-typestarchesaremorepressure-resistantthanthoseoftheAandC types,butduetooverlaps,acleardistinctioncannotbemadeinthisregard(Rubensetal.,1999;Katopoetal.,2002).X-raystructure analysesalsoshowedthatanX-raydiffractionpatternchangeduringultra-high-pressuretreatmentcanoccur.Forexample,nativeA- typestarchesshowtheX-raydiffractionpatternofB-typestarchesaftertreatment,withthelatterundergoingnochange(Hibietal., 1993;Katopoetal.,2002).Accordingtotheauthorsmentioned,thischangeisduetothestructureoftheamylopectin.Asalready mentioned, B-type starches have channels between the double helices in which a large number of water molecules can be embedded.Underultra-high-pressureconditions,theseinteractwithandstabilizethedoublehelices.Duetothemolecularflexi- bilityoftheA-typedoublehelices,thesecanformchannelsunderultra-high-pressureconditions.Incombinationwiththethermal process,gelatinizationunderultra-highpressurecanbeachievedbelowtheatmosphericgelatinizationtemperaturesinceultra-high pressurelowersit.Asthetemperaturerises,thispressureeffectbecomesweaker(Buckowetal.,2007). Thefeatureshighpressuretreatedstarchofdifferentplant-basedoriginsofferarenotfullyassessedandused.Onepossibility couldbetousethetexturetodesignandbuildlowfatfoodssincethegelshaveamouthfeellikefat.Furtherstarchincombinations withe.g.proteinscouldbeusedtocreateahybridnetworksofstarchandproteinstodevelopmeatfreeplant-basedproteinrichand fiberrichfoods.Duetothenatureofthestarchafterthetreatment,thestarchwouldberesistantandthereforethefoodwouldhave alowglycemicindex.Papathanasiouetal.(2015)showedthat5%starchsolutionsofwheat,tapioca,potato,corn,waxycornand resistantstarch(RS3)releasedlessglucoseafter120minofenzymaticdigestionsiftheywerepressurizedat600MPafor15minat roomtemperatureincomparisontoheattreatedstarchsolutions. 1.01.4.2 Pectin Pectinismainlyfoundinplant-basedproducts.Herethehighestamountsarefoundinapple(1.5%w/w)andcarrots(1.4%w/w). Inprocessing,pectin,whichismainlyfoundinthemiddlelamella,canbealteredbyeitherchemicalorbiochemicalconversions. Thiscanbeinfavorofthetexture(demethoxylation)ordetrimental(depolymerization/beta-elimination)(VanDerPlanckenetal., 2012).Demethoxylationofpectincaneitherbetriggeredbynon-enzymaticorenzymaticdemethoxylation(Pectinmethylesterase, PME).Inthecaseofhigh-pressureprocessing,bothmechanismscouldbepresentsimultaneouslysincetheprocessdoesnotaffect PMEtosuchanextentthatitwouldbefullyinactivated.Therateofthisreactionisacceleratedwithincreasingdegreesofmethyl- ation,temperature,andpH(4–6)(Diazetal.,2007).EspeciallythepHforfruitandvegetable-basedproductsisatanoptimum.For non-enzymatic degradation of pectin there are two mechanisms that could apply:beta-elimination or demethoxylation by acid hydrolysis (Van Der Plancken et al., 2012; Chen et al., 2015). The ß-elimination reaction is primarily base-catalyzed, but can alsobecomedominantatpH>4,andleadstothecleavageataglycosidiclinkagenexttoanesterifiedgalacturonicacid;asaresult, pectinwithahighdegreeofmethoxylation(DM)ismoresubjecttoß-eliminationthanpectinwithalowDM(Chenetal.,2015).At lowdegreeofmethoxylation(DM),demethoxylationcanalsooccurbyacidhydrolysis,enhancedbytemperaturebutnotbypres- sure.TwopossibleexplanationsweresuggestedfortheimprovedretentionofhardnessunderpressurebyVanderPlanckenetal.