1994 colorado protein stability conference dr. ted labuza the maillard reaction...

1page

1994 Colorado Protein Stability ConferenceDr. Ted Labuza The Maillard Reaction of Sugars with Proteins

The Maillard ReactionThe Maillard Reaction

T.P.LabuzaT.P.Labuza University of Minnesota

Kinetics and applications


T.P.LabuzaT.P.Labuza

Ling 1908 work on beer

Brewing process browning Color from sugar & protein

Maillard 1912 model system

1:4 glycine + glucose @ 37¡C color & carbon dioxide

Ling 1908 work on beer

Brewing process browning Color from sugar & protein

Maillard 1912 model system

1:4 glycine + glucose @ 37¡C color & carbon dioxide



vvFour pathwaysFour pathwaysÐÐ reducing sugar + aminereducing sugar + amine

ÐÐ high temperaturehigh temperature carmelization carmelization (sugar oxidation) (sugar oxidation)

ÐÐ ascorbic acid (low pH juices)ascorbic acid (low pH juices)

ÐÐ lipidlipid peroxidation peroxidation

2page


ALDOSESUGAR +

AMINOCOMPOUND N-SUBSTITUTED

GLYCOSYLAMINE+

MAILLARD BROWNING PATHWAY

SCHIFF BASEOF HMF ORFURFURAL

AMADORI REARRANGMENT

REDUCTONES

(acidic)(basic)

M E L A N O I D I N S (BROWN NITROGENOUS POLYMERS

AND COPOLYMERS

-AMINO COMP' D + H2O -2H +2H

HMF OR FURFURAL

DEHYDRO REDUCTONES

(High T)

+ a-AMINO ACID

STRECKER DEGRADATION

CO2

+ AMINOCOMPOUND

ALDIMINES

+

FISSION PRODUCTS (ACETOL,PYRUVALDEHYDEDIACETYL, ETC)

WITH ORWITHOUT

AMINO COMP'D

H O2

-2 H2O

ALDOLS ANDN-FREE POLYMERS

+ AMINO COMP'D

ALDIMINESOR

KETIMINES

ALDEHYDE

+ AMINO COMP'D

ALDIMINES+ AMINO COMP'D

1-AMINO-1-DEOXY-2-KETOSE (1,2-ENOL FORM)

T ime

Relat

ive C

once

ntrat

ion

Fluoresence

Brown Pigment

ReducingSugar

Reducing Intermediate

A mine

AMINE

Fluorescence

Brown pigment

ReducingIntermediate

ReducingSugar

Maillard Reaction

Sequence

Maillard Reaction

Sequence



vv> 2000 types of confectioneries> 2000 types of confectioneries

vv thousands of recipesthousands of recipes

vvnon-chocolate productsnon-chocolate productsÐÐ main ingredient - sweetenersmain ingredient - sweeteners

ÐÐ most are reactive ( sucrose, sorbitol)most are reactive ( sucrose, sorbitol)

ÐÐ most are heated to some boiling temperaturemost are heated to some boiling temperature

ÐÐ final moisture determines state (glass vsfinal moisture determines state (glass vsrubbery)rubbery)

ÐÐ caramels have added reaction productscaramels have added reaction products

3page




vvUndesirable ConsequencesUndesirable ConsequencesÐÐ darkeningdarkening

ÐÐ loss of solubilityloss of solubility

ÐÐ loss of protein biological valueloss of protein biological value

ÐÐ toxicity/toxicity/mutagenicitymutagenicity (IQs) (IQs)



vvDesirableDesirable Consequenses ConsequensesÐÐdesirable flavors- eg cereal (indication of loss ofdesirable flavors- eg cereal (indication of loss oftoxicity) beer , coffee, caramels, chocolate, teatoxicity) beer , coffee, caramels, chocolate, tea

ÐÐincreased palatability/nutrition - indicator of donenessincreased palatability/nutrition - indicator of doneness

ÐÐbrown colors - toasting, roasting, chocolate, coffee,brown colors - toasting, roasting, chocolate, coffee,beerbeer

ÐÐgeneration of antioxidants/generation of antioxidants/ anticarcinogens anticarcinogens



Amadori productSchiff Base

2CO

-1

+F

sk

Strecker Degradation

BAk

4k

bk

[B] brown pigment

F

3k

2k

k '

1k

A + R*

ARRA*R + A

4page




Substrates: free amino group plus aldehydo or ketose group

[A] [R]

amino acids reducing sugars proteins-e-lysine ascorbic acid aspartame ketones disodium guanylate aldehydes MSG orthophenolics sucrose (hydrolyzed)



vvSweetener IngredientsSweetener IngredientsÐÐ relative sweetnessrelative sweetness

ÐÐ solubility and crystallizationsolubility and crystallization

ÐÐ hygroscopicityhygroscopicity

ÐÐ flavor developmentflavor development

vvSucrose hydrolysisSucrose hydrolysisÐÐ low pHlow pH

ÐÐ high temperaturehigh temperature

ÐÐ yields glucose + fructoseyields glucose + fructose



vvMajor factors in color/flavorMajor factors in color/flavorÐÐ time temperature sequencetime temperature sequence

ÐÐ water content profilewater content profile

ÐÐ type of reactantstype of reactants

°F

time

5page




− ddAAddtt

= − ddRRddtt

= kk1 AA[ ] RR[ ] − kk3 AARR[ ]Assume k 1 >>> k3



RatedRdt

k A R k R

R R eok t

= − = − [ ][ ] = − [ ]

= −

'

'


T.P.LabuzaT.P.LabuzaTime hours

% G

luco

se R

emai

ning

10

100

0 500 1000 1500 2000

0.05 M glycine0.2 M glycine0.4 M glycine

Baisier & Labuza JFS 40:707 (1992)

0.4 M glucose 37°C pH 7

k0.05 = 1.62 hr -1

k0.2 =3.62 hr -1

k0.4 = 7.78 h r-1

k1= 22 hr -1 M-1

6page




pH EffectspH Effects



Keq =RNH2[ ] H+[ ]RNH3

+[ ]@RNH2 = RNH3

+ logKeq = log H+[ ] = −pH = −pKa

∴% unprotinated =(10 pH - pKa )1001 + 10 pH - pKa



% Unprotinated

pH glycine Lysine

1 2.5 x 10-7 2.45x10-6

3 2.5 x 10-5 2.45x10-4

5 2.5 x 10-3 2.45x10-2

7 0.25 2.45

8.9 17 50

9 20 80

9.6 50 84

11 96 ~100

% Unprotinated

pH glycine Lysine

1 2.5 x 10-7 2.45x10-6

3 2.5 x 10-5 2.45x10-4

5 2.5 x 10-3 2.45x10-2

7 0.25 2.45

8.9 17 50

9 20 80

9.6 50 84

11 96 ~100

7page




acid region

log (kobs) = c + log[H+] = c - pH

slope = 1

10 fold decrease for 1 pH unit

basic region

log (kobs) = c +pOH=c+pH

slope =1

acid region

log (kobs) = c + log[H+] = c - pH

slope = 1

10 fold decrease for 1 pH unit

basic region

log (kobs) = c +pOH=c+pH

slope =1



pH

AA

k-1

(m

in^-

1) a

fter

8hr

@10

0°C

0.001

0.01

0.1

2 3 4 5 6 7 8 9 10 11 12 13 14

trytophanmethionine

Pilkova et al Die Nahrung 34:759 (1990)

pK=9.39

pK=9.2



problems with most studies

single points in time

high temperature changes with time

pH not controlled- usually drops ->decreases rate

amine - sugar ratio unknown and varies

moisture - time profimle unknown

problems with most studies

single points in time

high temperature changes with time

pH not controlled- usually drops ->decreases rate

amine - sugar ratio unknown and varies

moisture - time profimle unknown

8page




Sugar TypeSugar Typealphaform

C=O

betaform

aldehydo or ketose

H

HO

HO

H

HH

CH2OH

H

OH

OH

O

H

HO

HO

H

HH

CH2OH

OH

H

OH

O



% acyclic as function of pH

Sugar pH 6.5 pH 7.0 pH 7.5

glucose 0.012 0.022 0.040

mannose 0.040 0.062 0.110

ribose 10 18.5 30

% acyclic as function of pH

Sugar pH 6.5 pH 7.0 pH 7.5

glucose 0.012 0.022 0.040

mannose 0.040 0.062 0.110

ribose 10 18.5 30

Cantor & Peniston JAOC 62:2112 (1940)



sugar k (10 -3 mM-1h-1) ratio to %acyclic ratio to glucose glucose

glucose 0.6 1 0.002 1 mannose 3.6 6 0.005 2.5 galact ose 2.8 4.7 0.02 10 ribose 10 16.7 0.05 25 fructose 4.5 7.5 0.7 350

sugar k (10 -3 mM-1h-1) ratio to %acyclic ratio to glucose glucose

glucose 0.6 1 0.002 1 mannose 3.6 6 0.005 2.5 galact ose 2.8 4.7 0.02 10 ribose 10 16.7 0.05 25 fructose 4.5 7.5 0.7 350

Bunn & Higgins Sci:213:222 (1981)

Sugar:Hemoglobin (5:1) pH 7.3 @ 37°C

9page




% acylic

k m

M-1

hr-

1

0.0001

0.001

0.01

0.1

0.001 0.01 0.1 1 10

aldehyoketo

Bunn & Higgins Sci:213:222 (1981)

Sugar:Hemoglobin (5:1) pH 7.3 @ 37°C



R1-NH2 + R2 - CHO --->Schiff Base

Schiff Base---->AR

AR--->Fluoresence

AR------->Pigments

R1-NH2 + R2 - CHO --->Schiff Base

Schiff Base---->AR

AR--->Fluoresence

AR------->Pigments



dd AARR[ ]ddtt

= kkAA RR[ ] − kk3 AARR[ ]= kkAA RRooee−kkAAtt[ ] − kk3 AARR[ ]

AARR[ ] =kkAA RRoo[ ]kk3 − kkAA

ee−kkAAtt − ee−kk3tt[ ]determine kA from initial R loss rate

non-linear regression to solve for k3

determine kA from initial R loss rate

non-linear regression to solve for k3

10page




Time hours

fluor

esce

nce

010002000300040005000600070008000

0 200 400 600



FF = kk3kkAA RR0[ ] ee−kkAAtt

kk3 − kkAA( ) kkBB − kkAA( ) + ee−kkAAtt

kkAA − kk3( ) kkBB − kk3( ) + ee−kkBBtt

kkAA − kkBB( ) kk3 − kkBB( )



Brown Pigment

Formation Rate

Brown PigmentBrown Pigment

Formation RateFormation Rate

11page




BB = RR0

+ BB0 + FF0[ ] 1− ee−kkBBtt[ ] − kk3kkAAkkBB RR0[ ]xxee−kk3tt

kk3 kkAA − kk3( ) kkBB − kk3( ) + ee−kkAAtt

kkAA kk3 − kkAA( ) kkBB − kkAA( ) + ee−kkBBtt

kkBB kk3 − kkBB( ) kkAA − kkBB( )



Time days

OD/gsolids

00.10.20.30.40.50.60.70.8

0 20 40 60

45 °C35 °C25 °C

Casein - glucose aw = 0.7



dd BB[ ]ddtt

= kkBB FF[ ] + kkAABB AA[ ] FF[ ] ≈ kkBB

BB = BBoo + kkBBtt

oorr BB = BBiinndd + kkBB ((tt −ttiinndd ))

Pseudo Zero Order

12page




Factors influencing browningFactors influencing browning

ratio and type of AA and sugarratio and type of AA and sugar

sugar: amine ratio sugar: amine ratio

pHpH

solvent statesolvent state

temperaturetemperature



Baisier data (pH 7 @ 37¡C with 0.1 M glycine)

glucose browning rate glucose browning

M OD/h x 104 ratio ratio

0.5 625 12.5 55

0.3 326 7.5 28.9

0.2 196 5.0 17.4

0.1 55 2.5 4.9

0.06 24 1.5 2.1

0.04 11 1 1

Baisier data (pH 7 @ 37¡C with 0.1 M glycine)

glucose browning rate glucose browning


0.5 625 12.5 55

0.3 326 7.5 28.9

0.2 196 5.0 17.4

0.1 55 2.5 4.9

0.06 24 1.5 2.1

0.04 11 1 1



Baisier data (pH 7 @ 37¡C with 0.4 M glucose)

glycine browning rate glycine browning


0.4 68 10 23

0.3 62 7.5 21

0.2 49 5.0 16

0.1 46 2.5 14

0.04 3 1 1

Baisier data (pH 7 @ 37¡C with 0.4 M glucose)

glycine browning rate glycine browning


0.4 68 10 23

0.3 62 7.5 21

0.2 49 5.0 16

0.1 46 2.5 14

0.04 3 1 1

13page




Time hours

OD

@49

0 nm

00.020.040.060.08

0.10.120.140.160.18

0.2

0 20 40 60 80

arginineglycinealanineproline

Wolfrom et al 1974 JAgFChem 22:791

1:1 M AA/Glucose@ 65°C

pk=9.09 pk=9.6

pk=9.87

pk=10.6



Massaro & Labuza JFS 55:821 (1993)

Ð model TPN solution @ 30¡C and pH 6.5

Lysine 0.008 OD/mole day

Tryptophan 0.259 OD/mole day

Cysteine 0.264 OD/mole day

L+T+C 0.054 OD/mole day

Commercial 0.019 OD/mole day

MassaroMassaro & Labuza JFS 55:821 (1993) & Labuza JFS 55:821 (1993)

ÐÐ model TPN solution @ 30¡C and pH 6.5model TPN solution @ 30¡C and pH 6.5

Lysine Lysine 0.008 OD/mole day 0.008 OD/mole day

TryptophanTryptophan 0.259 OD/mole day 0.259 OD/mole day

CysteineCysteine 0.264 OD/mole day 0.264 OD/mole day

L+T+C 0.054 OD/mole dayL+T+C 0.054 OD/mole day

Commercial 0.019 OD/mole dayCommercial 0.019 OD/mole day



pH

OD

@42

0 nm

0

0.2

0.4

0.6

0.8

1

1.2

6 8 10 12

alaninelysinearginine

Ashoor & Zent JFS 4:1206 1984

10 hr @ 120°C

pK=9.09

pK=8.9

pK=9.87

14page




Rate

pH

1 3 5 7 9pH

General Protein Browning



SchnicklesSchnickles et al 1976 J et al 1976 J Ag Ag F Chem 24:901 F Chem 24:901

Protein lysine OD/g solids day OD/mgProtein lysine OD/g solids day OD/mg

(mg) lysine day (mg) lysine day

gluten +gluten +

10% 10% lys lys 33 34 1.1 33 34 1.1

casein 14 10 0.7casein 14 10 0.7

whey 8.6 8.5 1.0whey 8.6 8.5 1.0

soy 7.6 7.5 1.0soy 7.6 7.5 1.0

eggegg alb alb 3.6 4.9 1.4 3.6 4.9 1.4



Sugar Gel Strength Color (L)

sucrose no gel nonelactose 125 77.6fructose 148 69.1mannose 193 64.5xylose 287 43.2

Mitchell U Nottingham - BSA + 3% sugar 60 min @ 121¡C

15page




vv Conditioning of potatoes for chipsConditioning of potatoes for chips

vv glandlessglandless cottonseed - cottonseed - gossypoll gossypoll

vv de-sugaring of eggs for dryingde-sugaring of eggs for drying

vv processing temperature controlprocessing temperature control

Industrial Control of Browning



Time days

OD/gsolids

00.10.20.30.40.50.60.70.8

0 20 40 60

45 °C35 °C25 °C

Casein - glucose aw = 0.7

Temperature EffectTemperature Effect



Item aw T¡C EA

(Kcal/mole)

dry cabbage 0.1 25-40 40

casein-glucose 0.5 25-45 33

processed cheese 0.97 5-40 24

glucose-glycine 1 60-100 20

16page




Product % blocked lysineraw milk 0freeze dried milk 0pasteurized milk 74°C 40 sec 0UHT pasteurized 140°C 3 sec 0-2spray dried milk 0-3sweetened condensed milk 8-12roller dried (no preconcentration) 10-15evapo rated milk 15-20drum dried 20-30

J. Mauron Nestle Co.

Nutritional Consequenses



Time

Relat

ive C

once

ntra

tion

Fluoresence

Brown Pigment

ReducingSugar

Reducing Intermediate

Amine

amine

reducing

sugar

Flouresence

Brown

Pigment

reducing

intermediates



vv decreased weight gaindecreased weight gain

vv increased liver weightincreased liver weight

vv increased kidney weightincreased kidney weight

vv increasedincreased ceacum ceacum

vv increased heart weightincreased heart weight

vv increased serum GOT, APincreased serum GOT, AP

vv accumulation of pigment in tissuesaccumulation of pigment in tissues

vv presencespresences of vacuolated of vacuolated hepatocytes hepatocytes

vv increased Zinc loss in urineincreased Zinc loss in urine

Toxicological Consequenses

eg Lee et al 1981 rats fed 10 months with browned casein 10% dietalso with 5-10% MRP in diet

17page




Toxicological/Nutritional consequences to humans

MRPs ~ 1.5-2% of diet

? of mineral retention

protein bioavailability

pro/anti mutagenicity of MRPs (imidiazo quinolines and furfural) especially heated foods

high fructose corn syrup in normal diet (1/3 into serum)



vv diabetes control -diabetes control - glycated glycated hemoglobin hemoglobin

vv atherosclerosisatherosclerosis initiation initiation

vv reduced elasticity of connective tissuereduced elasticity of connective tissue

vv nephropathynephropathy

vv kidney function - basement membranekidney function - basement membrane

vv retinopathy eg corneal protein - cataractsretinopathy eg corneal protein - cataracts

Medical Aspects



vvGlycosylationGlycosylation inhibitors inhibitors

ÐÐtoxicity of sulfite - use intoxicity of sulfite - use inprocessingprocessing

ÐÐteatea catechins catechins

ÐÐdrugsdrugs

Medical Aspects

18page




? ?

AnyQuestions

University of Minnesota

1994 colorado protein stability conference dr. ted labuza the maillard reaction...

Documents