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
The Maillard ReactionThe Maillard Reaction
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
The Maillard ReactionThe Maillard Reaction
T.P.LabuzaT.P.Labuza
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
1994 Colorado Protein Stability ConferenceDr. Ted Labuza The Maillard Reaction of Sugars with Proteins
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
The Maillard ReactionThe Maillard Reaction
T.P.LabuzaT.P.Labuza
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
1994 Colorado Protein Stability ConferenceDr. Ted Labuza The Maillard Reaction of Sugars with Proteins
The Maillard ReactionThe Maillard Reaction
T.P.LabuzaT.P.Labuza
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)
The Maillard ReactionThe Maillard Reaction
T.P.LabuzaT.P.Labuza
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
The Maillard ReactionThe Maillard Reaction
T.P.LabuzaT.P.Labuza
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
1994 Colorado Protein Stability ConferenceDr. Ted Labuza The Maillard Reaction of Sugars with Proteins
The Maillard ReactionThe Maillard Reaction
T.P.LabuzaT.P.Labuza
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)
The Maillard ReactionThe Maillard Reaction
T.P.LabuzaT.P.Labuza
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
The Maillard ReactionThe Maillard Reaction
T.P.LabuzaT.P.Labuza
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
1994 Colorado Protein Stability ConferenceDr. Ted Labuza The Maillard Reaction of Sugars with Proteins
The Maillard ReactionThe Maillard Reaction
T.P.LabuzaT.P.Labuza
− ddAAddtt
= − ddRRddtt
= kk1 AA[ ] RR[ ] − kk3 AARR[ ]Assume k 1 >>> k3
The Maillard ReactionThe Maillard Reaction
T.P.LabuzaT.P.Labuza
RatedRdt
k A R k R
R R eok t
= − = − [ ][ ] = − [ ]
= −
'
'
The Maillard ReactionThe Maillard Reaction
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
1994 Colorado Protein Stability ConferenceDr. Ted Labuza The Maillard Reaction of Sugars with Proteins
The Maillard ReactionThe Maillard Reaction
T.P.LabuzaT.P.Labuza
pH EffectspH Effects
The Maillard ReactionThe Maillard Reaction
T.P.LabuzaT.P.Labuza
Keq =RNH2[ ] H+[ ]RNH3
+[ ]@RNH2 = RNH3
+ logKeq = log H+[ ] = −pH = −pKa
∴% unprotinated =(10 pH - pKa )1001 + 10 pH - pKa
The Maillard ReactionThe Maillard Reaction
T.P.LabuzaT.P.Labuza
% 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
1994 Colorado Protein Stability ConferenceDr. Ted Labuza The Maillard Reaction of Sugars with Proteins
The Maillard ReactionThe Maillard Reaction
T.P.LabuzaT.P.Labuza
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
The Maillard ReactionThe Maillard Reaction
T.P.LabuzaT.P.Labuza
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
The Maillard ReactionThe Maillard Reaction
T.P.LabuzaT.P.Labuza
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
1994 Colorado Protein Stability ConferenceDr. Ted Labuza The Maillard Reaction of Sugars with Proteins
The Maillard ReactionThe Maillard Reaction
T.P.LabuzaT.P.Labuza
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
The Maillard ReactionThe Maillard Reaction
T.P.LabuzaT.P.Labuza
% 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)
The Maillard ReactionThe Maillard Reaction
T.P.LabuzaT.P.Labuza
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
1994 Colorado Protein Stability ConferenceDr. Ted Labuza The Maillard Reaction of Sugars with Proteins
The Maillard ReactionThe Maillard Reaction
T.P.LabuzaT.P.Labuza
% 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
The Maillard ReactionThe Maillard Reaction
T.P.LabuzaT.P.Labuza
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
The Maillard ReactionThe Maillard Reaction
T.P.LabuzaT.P.Labuza
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
1994 Colorado Protein Stability ConferenceDr. Ted Labuza The Maillard Reaction of Sugars with Proteins
The Maillard ReactionThe Maillard Reaction
T.P.LabuzaT.P.Labuza
Time hours
fluor
esce
nce
010002000300040005000600070008000
0 200 400 600
The Maillard ReactionThe Maillard Reaction
T.P.LabuzaT.P.Labuza
FF = kk3kkAA RR0[ ] ee−kkAAtt
kk3 − kkAA( ) kkBB − kkAA( ) + ee−kkAAtt
kkAA − kk3( ) kkBB − kk3( ) + ee−kkBBtt
kkAA − kkBB( ) kk3 − kkBB( )
The Maillard ReactionThe Maillard Reaction
T.P.LabuzaT.P.Labuza
Brown Pigment
Formation Rate
Brown PigmentBrown Pigment
Formation RateFormation Rate
11page
1994 Colorado Protein Stability ConferenceDr. Ted Labuza The Maillard Reaction of Sugars with Proteins
The Maillard ReactionThe Maillard Reaction
T.P.LabuzaT.P.Labuza
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( )
The Maillard ReactionThe Maillard Reaction
T.P.LabuzaT.P.Labuza
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
The Maillard ReactionThe Maillard Reaction
T.P.LabuzaT.P.Labuza
dd BB[ ]ddtt
= kkBB FF[ ] + kkAABB AA[ ] FF[ ] ≈ kkBB
BB = BBoo + kkBBtt
oorr BB = BBiinndd + kkBB ((tt −ttiinndd ))
Pseudo Zero Order
12page
1994 Colorado Protein Stability ConferenceDr. Ted Labuza The Maillard Reaction of Sugars with Proteins
The Maillard ReactionThe Maillard Reaction
T.P.LabuzaT.P.Labuza
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
The Maillard ReactionThe Maillard Reaction
T.P.LabuzaT.P.Labuza
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
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
The Maillard ReactionThe Maillard Reaction
T.P.LabuzaT.P.Labuza
Baisier data (pH 7 @ 37¡C with 0.4 M glucose)
glycine browning rate glycine browning
M OD/h x 104 ratio ratio
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
M OD/h x 104 ratio ratio
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
1994 Colorado Protein Stability ConferenceDr. Ted Labuza The Maillard Reaction of Sugars with Proteins
The Maillard ReactionThe Maillard Reaction
T.P.LabuzaT.P.Labuza
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
The Maillard ReactionThe Maillard Reaction
T.P.LabuzaT.P.Labuza
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
The Maillard ReactionThe Maillard Reaction
T.P.LabuzaT.P.Labuza
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
1994 Colorado Protein Stability ConferenceDr. Ted Labuza The Maillard Reaction of Sugars with Proteins
The Maillard ReactionThe Maillard Reaction
T.P.LabuzaT.P.Labuza
Rate
pH
1 3 5 7 9pH
General Protein Browning
The Maillard ReactionThe Maillard Reaction
T.P.LabuzaT.P.Labuza
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
The Maillard ReactionThe Maillard Reaction
T.P.LabuzaT.P.Labuza
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
1994 Colorado Protein Stability ConferenceDr. Ted Labuza The Maillard Reaction of Sugars with Proteins
The Maillard ReactionThe Maillard Reaction
T.P.LabuzaT.P.Labuza
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
The Maillard ReactionThe Maillard Reaction
T.P.LabuzaT.P.Labuza
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
The Maillard ReactionThe Maillard Reaction
T.P.LabuzaT.P.Labuza
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
1994 Colorado Protein Stability ConferenceDr. Ted Labuza The Maillard Reaction of Sugars with Proteins
The Maillard ReactionThe Maillard Reaction
T.P.LabuzaT.P.Labuza
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
The Maillard ReactionThe Maillard Reaction
T.P.LabuzaT.P.Labuza
Time
Relat
ive C
once
ntra
tion
Fluoresence
Brown Pigment
ReducingSugar
Reducing Intermediate
Amine
amine
reducing
sugar
Flouresence
Brown
Pigment
reducing
intermediates
The Maillard ReactionThe Maillard Reaction
T.P.LabuzaT.P.Labuza
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
1994 Colorado Protein Stability ConferenceDr. Ted Labuza The Maillard Reaction of Sugars with Proteins
The Maillard ReactionThe Maillard Reaction
T.P.LabuzaT.P.Labuza
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)
The Maillard ReactionThe Maillard Reaction
T.P.LabuzaT.P.Labuza
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
The Maillard ReactionThe Maillard Reaction
T.P.LabuzaT.P.Labuza
vvGlycosylationGlycosylation inhibitors inhibitors
ÐÐtoxicity of sulfite - use intoxicity of sulfite - use inprocessingprocessing
ÐÐteatea catechins catechins
ÐÐdrugsdrugs
Medical Aspects