A New Acid Stable Thermostable Maltogenic …

1 Duan Gang, Qian YingMartjin ScheffersGerhard Konieczny-JandaJay ShettyLeidenGenencor, DaniscoA New acid Stable Thermostable Maltogenic alpha amylase for High Maltose Syrup58 Detmold Starch Convention, Germany2 AgendaCurrent maltose production and applicationThe issueWhat is the new enzyme?Application dataSummary3 Maltose ApplicationApplicationsFunctionsBaking Good heat stability Reduced browningBrewing High fermentabilityCandy Low hygroscopicity Resists crystallizationCanning Low sweetnessSoft drinks Body/flavor characteristics Pleasant mouth feelFood formulations Good heat stability Low hygroscopicity Low viscosityHydrogenated maltose (maltitol) Low calorie sweetenerNutrient (Japan) Intravenous feedingHealth (bifidobacteria) and functional food ingredients Substrate for IMO sugars4 Typical Process of Maltose ProductionStarch ( Maize or Tapioca or other)(15-32% dsb)Liquefaction(DE: 4-11) alpha amylase Inactivation(pH , 30 min.)

2 At 95 oC, or second jet at 120+ C) alpha amylase (SPEZYME FRED or SPEZYME AA)Saccharification(pH , 55-60 oC)Refining and finishDepending on original DE some residual starch mayremain that can be removed with alpha AA (CLARASE(R) L) - amylase ( OPTIMALT BBA)Pullulanase (OPTIMAX -L 1000)5 Beta-amylasePullulanaseMaltoseREACTION MECHANISM of BETA amylase ,PULLULANASE and FUNGAL alpha AMYLASEF ungal alpha Amylase6 Maltogenic Enzymes Sugar profiles Impact of DE Impact of pH and Temperature Impact of residual thermal Stable and pH Stable liquefaction enzyme Trouble shootingFactors Affecting the Production of Very High Maltose Syrup7 Analytic MethodAnalytical Methods for Carbohydrate AnalysisRecommended ConditionsHPLC columnHPX 87-H (Bio-Rad)Column temperature60 CMobile N H2SO4 DetectionRIInjection volume20 l, solutionmin024681012141618nRIU0100002000 03000040000500006000070000*RID1 A, Refractive Index Signal (2006\ )Blue is AKAA performanceRed is EDS 224 performance*RID1 A, Refractive Index Signal (2007\ )8 Sugar profileSugar Composition (%)Syrup TypeMaltoseGlucoseMaltose Syrup40 - 451 - 10 Fungal alpha - amylase CLARASE (R)-LHigh Maltose Syrup55 -601 -5 Beta- amylase OPTIMALT BBAVery High Maltose Syrup> 80< 1 Beta- amylase + pullulanaseOPTIMALT BBA + OPTIMAX Recommended Enzyme(s)9 The issue of the current process-pH DropDAMAGING MICROBE LACTOBACILLUS (fermentum)VERY Thermostable UP TO 60 oCPOPULATION GROWTH 52-56 oCFAST acid FORMATION CAPABILITYFACULTATIVE ANAEROBIC GROWTH10 What the industry do to deal with the issueoCleaning with/without chemical from time to time!

3 WASHING PROCESS EQUIPMENTS WITH HOTapp. 60oC, 1% NaOH, rinsing!WASHING AGAIN WITH HOT appr. 60 oC, HNO3( Note: Materials of tanks etc.), rinsing!FINALLY RINSING WITH COOL H2O2!REGULAR CLEANING (Own schedule must be created.)oShort reaction timeoAdd lysozyme11 New Fungal AA from Trichoderma reeseiSample: alpha amylase [SCAFFOLD 3]Conversion:Endohydrolysis of 1,4- -D-glucosidic linkages in polysaccharides containing three or more 1,4- -linked D-glucose :Starch degradation in bread baking, brewing, dishwashing and sweeteners and bioethanol applicationsDescription:It is an overexpressed enzyme produced by Trichoderma s new, TrAA, a low pH FAApH profile, TRAA, 13 Enzyme used in this studyClarase (R)-L : Fungal alpha amylase standard (#107-04330-001) 40690 SKBU/gOptimax L-1000 acid Stable pullulanase standard (#107-04224-001) 1036 ASPU/gSPEZYME FredLiquefact Spezyme Fred: Low pH Thermostable AA DE -10 TrAA-sample(150906).

4 17865 SKBU/g14 Dose response of TrAA for maltose Enzyme 1 (dose)Enzyme 2 (dose)T ( C)pHTime (hr)% DP1% DP2% DP3% (20 SKBU/g)PU ( kg/mt) (20 SKBU/g) (15 SKBU/g) (10 SKBU/g) of TrAA with FAA and BBAat low pHEnzyme (dose)T ( C)pHTime (hr)% DP1% DP2% DP3% ( kg/mtds) L( kg/mt ds) ( kg/mtds) of CLARASE and TrAA on maltose formation at higher temperature Sugar profile at 65C0102030405060DP1DP2DP3DP4DP4+DEDPs% of sugarTrAACLRASE(R)-LTemperature: 65 C; pH: of enzymes: TrAA: ; CLARASE(R)-L: time: 24 hrs 17 Effect of added Pullulanase on Maltose levelTrAAPUT ( C)pHTime (hr)% DP1% DP2% DP3% kg/mt kg/mt kg/mt kg/mt kg/mt kg/mt chart of the maltose processStarch SlurryStarch SlurrySolids 20 Solids 20 35%35%Jet CookerJet CookerDextrinationDextrinationDE 1 DE 1 -- 1010pH adjustmentpH adjustmentSaccharificationSaccharificati onHeating 70 Heating 70 80 C80 CBacterial alphaBacterial alpha --amylaseamylaseFungal Fungal alphaalpha--amylaseamylaseBetaBeta--amyl ase/ amylase / pullulanasepullulanaseFiltrationFiltrati onCarbon treatmentCarbon treatmentPolish filterPolish filterEvaporationEvaporation6060--70% ds70% dsDemineralize by Demineralize by ion exchangeion exchangeOptional pH adjustment Optional pH adjustment & Destarching ( amylase )& Destarching ( amylase )

5 BetaBeta--amylaseamylaseStarch SlurryStarch SlurrySolids 20 Solids 20 35%35%Jet CookerJet CookerDextrinationDextrinationDE 1 DE 1 -- 1010pH adjustmentpH adjustmentSaccharificationSaccharificati onHeating 70 Heating 70 80 C80 CBacterial alphaBacterial alpha --amylaseamylaseFungal Fungal alphaalpha--amylaseamylaseBetaBeta--amyl ase/ amylase / pullulanasepullulanaseFiltrationFiltrati onCarbon treatmentCarbon treatmentPolish filterPolish filterEvaporationEvaporation6060--70% ds70% dsDemineralize by Demineralize by ion exchangeion exchangeOptional pH adjustment Optional pH adjustment & Destarching ( amylase )& Destarching ( amylase )BetaBeta--amylaseamylase19 Benefits of using the new enzymeNo pH drop during SaccharificationLess contamination, less color substanceNo need for pH adjustment after AA deactivation 20 SummaryWith the new TrAA, saccharification of liquefied starch for maltose production could be done at low pH , or higher temperature, under which the microbial contamination issue will be suppressed.

6 The will offer operational benefits as no pH adjustments after deactivation of alpha - amylase and during saccharification, less cleaning of the reaction van SolingenYanping LiMarga OpdamVictor Vahanian22 New Facilitiesin Wuxi, China