Enzyme ersetzen E-Nummern & Energie

Dr. Lutz PopperSt E G bH & C KG Ah bSternEnzym GmbH & Co. KG, Ahrensburg

11. FEI Kooperationsforum 2012: Enzyme in der Lebensmittelproduktion: Neue Wege zur

1

p gGewinnung und Nutzung. Bonn, 17.04.2012

Enzymvielfalty

ca. 10.000 Enzyme in der Natur

ca. 100 Enzyme mit sich unterscheidenden katalytischen Aktivitäten werden fürkatalytischen Aktivitäten werden für technische Zwecke hergestellt

4 LP18052011

Enzyme Production: from Natural Sources only

MicrobesYeasts moulds bacteria

PlantsAnimals

Fermentation

Yeasts, moulds, bacteria

CultivationFarming

SETissue Cells BrothFluids,

e.g. rubberTissueFluids,

e.g. milk

SE

Disintegration

Aqueous extractionAqueous extraction

Filtration/separation

Concentration

Drying

5 LP22052007

Standardization/blending

Gründe zum Einsatz von Enzymen bei der L b itt l b itLebensmittelverarbeitung

Verbesserung der VerarbeitungSt kt i h f ti T b k it Strukturerweichung, -festigung, Trennbarkeit, Zusammenhalt, Sedimentation, Klärung

Qualitätsverbesserungg Stabilität, Aussehen & Struktur, Haltbarkeit, Geschmack

KostenersparnisA b t E i Z it R h t ff Ausbeute, Energie, Zeit, Rohstoffe

Deklarationsfreiheit Alternative zu ZusatzstoffenAlternative zu Zusatzstoffen

Problemlösung Acrylamid, Fehlgeschmack, Lactose-Intoleranz,

(Gluten-Unverträglichkeit, Allergene, Kontaminanten)Innovation

Getreide /Sojamilch

6

– Getreide-/Sojamilch– Lösliche Ballaststoffe

LP12042012

Enzymes in Food Applications, Examplesy pp , pApplication Enzyme examples PurposeB ki A l l t V l i h lf lifBaking Amylase, xylanase, protease

esterase, lipoxygenase, oxidaseVolume, processing, shelf-life,bleaching, dough stabilization

Brewing Amylase, glucanase, protease Fermentation, stabilityCheese Protease, lipase, lysozyme Structure, flavor, preservationConfectionery Invertase Structure, shelf-lifeEgg Glucose-oxidase phospholipase Oxygen removal whippingEgg Glucose oxidase, phospholipase Oxygen removal, whipping

properties, heat stabilityFlavors Lipase, lipoxygenase, ADH FFA, aldehydesFruit & vegetables Pectinase PME Softening firmingFruit & vegetables Pectinase, PME Softening, firmingJuices Pectinase, arabinase, amylase,

proteaseYield, clarification, stabilization

Lipids Lipase, phospholipase Transesterification, hydrolysisMeat & fish Protease, transglutaminase Softening, firmingWine Pectinase, protease, laccase Clarif., stabilization, flavor,

7 LP11042012

premoval of off-flavors

Enzymes are Processing Aids!y g

Processing aids are substancesProcessing aids are substancespresent in insignificant amounts butwithout function in the final food, or,removed from the food during processing

Enzymes are processing aids, with the exception ofLysozyme (preservative, E 1105)Invertase (softener, wetting agent, humectant, E 1103)

Australia & New Zealand have additional approvals for enzymes as additives:Am lases (flo r treatment agent A 1100)Amylases (flour treatment agent, A 1100)Glucose oxidase (anti-oxidant, A 1102)Lipases (flavour enhancers A 1104)

8

Lipases (flavour enhancers, A 1104)

LP14072009

Typical Concentration Range of Baking E C d t E l ifiEnzymes as Compared to Emulsifiers

Substance Conc (g/ton) Calculated onSubstance Conc. (g/ton) Calculated onEnzymes

alpha-Amylase 1 – 5 Wheat flourp yXylanase 1 – 8 Wheat flourCarboxyl esterase 0.5 – 10 Wheat flour

EmulsifiersLecithin 500 – 3,000 Wheat flourM /Di l id 1 000 10 000 Wh t flMono/Diglycerides 1,000 – 10,000 Wheat flourDatem 1,000 – 4,000 Wheat flour

(Contaminant guidelines)*(Contaminant guidelines)Lead (Pb) 0.3 WheatCadmium (Cd) 0.1 Wheat

10 LP19092011

*German Federal Agency for Consumer Protection and Food Safety

Enzymes Suggested for Bread and Flour Improvers

Enzyme Claimed Effect-Amylase, fungal Energy supply for yeast-Amylase, bacterial Liquefaction-Amylase, intermediate heat stable Anti-stalingAmyloglucosidase (glucoamylase) Energy supply, colour, flavourBranching enzyme (glucotransferase) Water bindingCellulase Water bindingFuranosidase, arabinofuranosidase Dough structure, water bindingFerulic & cumaric acid esterase Dough structure, water bindingGlutathion oxidase Protein strengtheningGlycolipase, galactolipase Dough stability & volume yieldß-Glucanase Structure, liquefactionGlucose / galactose / hexose oxidase Protein strengtheningHemicellulase, xylanase, pentosanase Dough structure, water binding, volume yieldLaccase, polyphenol oxidase Dough strengtheningLipase Flavour, emulsification, dough stability & vol. yieldLipoxygenase, lipoxidase Dough structure, decolorizationexo-Peptidase Colour, flavourP id P i h iPeroxidase Protein strengtheningPhospholipase Pore structure & volume yieldProtease, proteinase Protein relaxation, liquefactionPullulanase Structure, water bindingS l hh d l id & t f P t i t th i

11 LP09062004

Sulphhydryl oxidase & transferase Protein strengtheningTransglutaminase Protein cross-linking, gluten stabilization

Carboxyl Esterases and Their Applications in F dFood

Enzyme Reaction Catalysed ApplicationsLipase (triacylglycerol lipase)

Splits fats and lipids into fatty acids and glycerol or other

Maturing of cheese; emulsifier production; interesterificationlipase) acids and glycerol or other

alcoholsproduction; interesterification of fats; baking

Phospholipase A2Phospholipase A1

Hydrolyses phospholipids (lecithin)

Improvement of emulsifying po er (e g egg olk)Phospholipase A1

Lyso-phospholipaseGalactolipase Splits fatty acids off Improvement of emulsifying

(lecithin) power (e.g. egg yolk); degumming, baking

galactolipids power; bakingAcetyl esterase Splits off acetyl groups, e.g.

from pectin or xylanBaking; fruit juice

from pectin or xylan

12 LP18052011

Classification and Distribution of the Main Lipids in Wheat Flour (% d s )Lipids in Wheat Flour (% d.s.)

Wheat flour lipids 1.4 - 2.0

Free lipids Bound lipids0 8 - 1 0 0 6 - 1 00.8 1.0 0.6 1.0

Nonpolar Polar Nonpolar Polar0 6 0 7 0 2 0 3 0 2 0 3 0 4 0 70.6 - 0.7 0.2 - 0.3 0.2 - 0.3 0.4 - 0.7

Glycolipids Phospholipids Glycolipids Phospholipids65 70 30 35 45 50 50 5565 - 70 30 - 35 45 - 50 50 - 55

13 LP27042011

Modif. from Pomeranz and Chung, 1978

Impact of Dough Mixing on Wheat Lipidsp g g p

• 2 – 2.8 % lipids in dry matter of flour, thereofp y ,• approx. 1 % bound lipids [1];• mostly phospholipids, bound to starch [2, 3];y p p p , [ , ];• during mixing, free polar lipids and triglycerides bind to

protein [4];• lipid oxidation by wheat lipoxygenase [5] • hydroperoxidesy p• oxidation of carotenoids (bleaching) and• oxidation of thiol groups (dough strengthening) [6]g p ( g g g) [ ]

[1] Chung, 1991; [2] Frazier et al., 1981; [3] Marion et al., 1987; [4] Mann & Morrison, 1974;[5] Tsen & Hlynka, 1962; [6] Nicolas & Drapon, 1983

14 LP18042011

[5] Tsen & Hlynka, 1962; [6] Nicolas & Drapon, 1983

Action of Carboxyl Esterases on Wheat Lipidsy p

Phospho-lipases

T i l liTriacyl lipases

(C & D = no carboxyl esterases)

Galacto-li

15 LP18052011

lipases

Effect of Wheat Lipids on Volume Yield of Defatted Wheat FlourDefatted Wheat Flour

polar lipids660ur]

s, 1

973

polar lipids660

0 g

flou

itchi

e &

Gra

s

total lipids600

[ml/1

00

f. fro

m M

acR

non-polar lipids530

olum

e

Mod

ifnon-polar lipids

460read

vo

volume prior to baking460Br

0 0.6 0.9 1.2 1.5 1.8 2.1 2.4 2.70.3 3.0

16 LP04072002

Re-added wheat lipid [g/100 g flour]

Production Scheme for Food Emulsifiers

Fats, oils Fatty acids

Sugar Glycerol Ethyleneoxide Sorbitol Polyglycerol Propylenel l Lactic acid

+ + + ++

Sugar Glycerol

Sugarglyceride

Mono-glyceride

Ethyleneoxide Sorbitol Polyglycerol glycol Lactic acid

Stearoyl-Polyglycerinester

Propyleneglycol ester

Sorbitan fattyacid ester

Poly-sorbateglyceride

E 474glyceride

E 471 lactic acidesterE 475

glycol esterE 477

acid esterE 491 - 495

sorbateE 432 - 436

+ ++

Mono- &diacetyl

tartaric acidLactic acid Citric acidAcetic acid

anhydrid Na CO3Ca(OH)

2 2

LACTEME 472 b

CITREME 472 c

ACETEME 472 a

DATEME 472 e

SSLE 481

CSLE 482

17 LP13032001

Production of Diacetyltartaric Acid Esters of Mono- and Diglycerides of Edible FatsMono- and Diglycerides of Edible Fats

Grapes Sugar cane, beets Fat, oil

Tartar

Wine Ethanol

Vinegar

Tartaric acid Acetic acid anhydride Glycerol Fatty acids

Diacetyltartaric acid Monoglycerides

Reaction vessel

ReactionReaction

Distillation

Cooling

Grinding

18

Powder packing

LP13042012

Microbial Enzyme Production Flow Diagramy g

Mixing PrecipitationWater bMixing

Sterilization

Precipitation

Ultrafiltration

Nutrients

SeparationaSterilization

Fermentation

Ultrafiltration

Dehydration

Microorganisms

p

b

Aqueous extraction1 Standardization Carrier

a

Separation Sieving

PackingMicrofiltration

19 LP16042012

1 only surface culture

Structure of Diacetyltartaric Acid Esters of Mono- and Diglycerides (DATEM)Mono- and Diglycerides (DATEM)

O

O

O

RO AcO

O OH

O R

HOO OH

AcO O

Ac = acetate residuec acetate es dueR = fatty acid residue, e.g. stearate

20 LP18092002

Rheofermentometer

21 LP19062008

Rheofermentometer Evaluation – Gas Release(c

m) 4

Hei

ght (

2

3

H

1

1 2 3Fermentation time (h)

H'm = maximum height of the gas release curve Retention coefficient =H m = maximum height of the gas release curveT1 = time to reach H'm

Tx = begin of gas leaking from the doughA1 = retention volume of CO2 in the dough

Retention coefficient = A1/(A1 + A2)·100

22 LP19062008

A1 retention volume of CO2 in the doughA2 = volume of CO2 lost from the dough

Replacement of DATEM by Alphamalt EFX M Rh f tMega - Rheofermenter

100

120

) Reference

60

80

ht (m

m)

EFX Mega 25 ppm + 0.12% DATEM

40

60

gh h

eig EFX Mega 50 ppm

DATEM 0.24%

0

20

Dou

0.0 0.5 1.0 1.5 2.0 2.5 3.0

Fermentation time (h)

23 LP25012011

Alphamalt EFX Mega – Breakfast Rolls from F D hFrozen Dough

800

850

flour

)

Reference

750

/100

g f common bread improver with

DATEM

650

700

eld

(mL/ common bread improver with

DATEM -50%, + 50 ppm EFX Mega

600

650

ume

yie

common bread improver with DATEM, -25% + 25 ppm EFX Mega

550Vol pp g

24 LP25012011

Effect of Dosage and Proof Time on Baguette Rolls with a Commercial Carboxyl EsteraseRolls with a Commercial Carboxyl Esterase

0 ppm 10 ppm 25 ppm 50 ppmBasic treatment:alpha amylase

1.5 h, normal proof

alpha-amylaseoxidizing agentsemulsifier

743 748 787 856

2 h, over-proof 1

Volume yield,mL/100 g flour p

803 852 882 935

2.5 h, over-proof 2

25

863 937 965 1015LP01062007

Steamed Bread with Triacyl Lipase LP 10066y p

Reference LP 10066, 5 ppm

300 mL / 100 g flour 447 mL / 100 g flour

LP 10066, 50 ppmLP 10066, 25 ppm

26 LP04072002

477 mL / 100 g flour 512 mL / 100 g flour

Steamed Bread with Lipolytic Enzymesp y y600

ur) Triacyl-Lipase

400

500

/100

g fl

ou

(Amylase) Galacto-Lipase

200

300

yiel

d (m

L/

100

200

Volu

me

y

0

V

27 LP09052007

Enzyme, dosage (ppm)

Bleaching Mechanism of Triacyl Lipaseg y p

Triglyceride FFA + lyso-lipidTriacyl Lipase(1)Triglyceride FFA lyso lipid

FFA + O2 R-OOH (hydro peroxide)Lipoxygenase(2)2 ( y p )

R-OOH bleaches the flour pigment and oxidizes the p gthiol groups of proteins

(1) Intrinsic or added triacyl lipase(2) Flour lipoxygenase type 1

TKP1708201128

Results of Southern China Steamed Bun Trials

Parameters Control C. Esterase Compound

Handling Normal Normal Slightly softerHandling Normal Normal Slightly softer

Crust colour L 87.36 89.19 89.48

Crust colour b 15.86 14.70 14.60

Crumb colour L 83.23 84.41 84.80

Crumb colour b 17.77 16.44 16.33

Specific volume 1.51 1.73 1.76

Skin appearance Wrinkles & collapse smooth smooth

29 TKP17082011

Aspects of Southern China Steamed Bun Trialsp

Control Esterase Compound

30 TKP17082011

Carboxyl Esterases Summaryy y

Carboxyl esterases act on lipids bound to or associated with proteinThe released lyso-forms of the lipids interact immediately

ith th t iwith the proteinThe gas retention capacity is improved rather than the shelf life of the crumb softnessshelf life of the crumb softnessThe steamed bread preparation process enhances the effect of triacyl lipases probably due to improved accesseffect of triacyl lipases, probably due to improved access of O2

Carboxyl esterases can reduce the use of emulsifiersCarboxyl esterases can reduce the use of emulsifiersSome carboxyl esterases support the flour’s own lipoxygenase in brightening of the crumb colourpo yge ase b g te g o t e c u b co ou

32 LP12042012

Improvement of Shelf-Life by Lipasep y p

Li l f tt id & di l id• Lipase releases fatty acids & di- or monoglycerides• potential to interact with starch, but

h li id l d i d i h i • the lipids are already associated with protein • intermediate interaction of FA and DG/MG with protein • no complexation in starch, no retardation of starch

retrogradation, buti t f i iti l b t t & b d l • improvement of initial crumb structure & bread volume

• improved crumb softness after storage

33 LP18042011

Shelf-Life Extension of White Tin Bread with Maltogenic Enzymes or EmulsifiersMaltogenic Enzymes or Emulsifiers

1900

1700

1900

1300

1500

e (g

)

Dosage (% on flour)

900

1100

Forc

e

Reference

F 9023, 0.03

500

700 Monoglyceride, 1.0

5001 5 7

Shelf life (days)

34

( y )

LP11042012

Ökonomische und ökologische Aspekte von E fü di B tf i hh ltEnzymen für die Brotfrischhaltung

Verlängerte Mindesthaltbarkeit abgepackter WareReduzierter Rücklauf von altbackener WareVerlängerte Serviceintervalle am RegalKonsumenten entsorgen weniger Endproduktg g p

Geringere Kosten für Produzent, Handel und Konsument Geringere Kosten für Produzent, Handel und Konsument Verringerter Carbon Footprint durch weniger Rohstoff-

und Kraftstoffverbrauch

35 LP11042012

Enzyme ersetzen Zusatzstoffey

Lysozym NitratCarboxylesterasen EmulgatorenCarboxylesterasen EmulgatorenLipasen, Lipoxygenasen BleichmittelO id O id i i lOxidasen OxidationsmittelProteasen Metabisulfit, CysteinTransglutaminase Phosphat, Hydrocolloide

36

Energiekosten-Einsparpotential beiW ff l

50 s 100

Waffelmassen

40

rspa

rnis

90

ewic

ht46

cm

)

20

30

oste

ner

(%)

70

80

ches

Ge

el, 2

9x4

10

20

ergi

eko

60

70

pezi

fisc

g/W

affe 12

0100120140160

En50

Sp (g

100120140160

Wassergehalt der Waffelmasse (kg/100 kg flour)

1 : mit LQ4020

2 h E

0,15 €/kWh0,11 €/kg Wasser( g g )

2 : ohne Enzym

LP1104201237

Enzyme sparen Energie – weitere Beispieley p g p

K ä k b t S lb ö l T k d lKnäckebrot, Semmelbrösel,Trockennudeln Verringerung der zu verdampfenden Wassermenge

BrennereiBrennerei Aufschluss der Rohstoffe bei 80 – 95 °C mit thermostabilen

Enzymen statt bei 130 °Cy

Herstellung von Glucosesirup Invertase bei 40 °C statt Säurehydrolyse bei 60-70 °C

Würzeherstellung Proteinhydrolye mit Proteasen und Ribonucleasen bei 40 °C statt

Sä h d l it HCl (4 bi 9 M) b i 70 135 oC fü 8 hSäurehydrolyse mit HCl (4 bis 9 M) bei 70-135 oC für 8 h, manchmal sogar 20-35 h

Keine Bildung von 3-MCPD (3-Monochloropropan-1,2-diol)g ( p p )

38 LP16042012

Energieeinsparung durch enzymatischeV i d W b tiVerringerung der Wasserabsorption

WaffelnKnäckebrotKnäckebrotSemmelbröselN d lNudelnBrauerei & Brennerei

39 LP11042012