The Role of pH in Maillard-Type Reactions 2004-09-08 NRC/FCI - IBk/Louvain Nestlé Research Center Outline of the presentation Basic principles of pH/Maillard • pH versus reactivity

Nestlé Research Center2004-09-08 NRC/FCI - IBk/Louvain1

The Role of pH in Maillard-Type Reactions

Chair J. de Clerck Symposium XILouvain, September 8, 2004

Imre BlankNestlé Research Center, Lausanne, Switzerland


Outline of the presentation

Basic principles of pH/Maillard

• pH versus reactivity (nucleophilicity)• pH versus buffer • pH to control the Maillard reaction

Examples from flavour research

• O-heterocycles – caramel-like/sweet• N-heterocycles – raosty/sweet• S-heterocycles – roasty/savoury


AromaTasteColour

AntioxidantsTexture

Nutritional valueContaminants

Toxic compounds

The first schemeof the Maillard reaction

(Hodge, 1953)


O

OHOH

OH

OH

OH

OH

OHOH

N

OH

OHCOOH OH

OOH

OH

OH

OHOH

NOH CH2

OH

NH

COOH

CH2

N

N

COOH

CH2

COOH

OH

OH

N

OOH

OHCOOHO

OH OHOH

COOHNOH

O

OH OHOH

CH2

N

O

OH OHOH

NH2

OH

OH

O

OOH

OH

OH

OH

NH

O

COOHOH

OH

O

NH

O

COOHOH

O

OHOH

NH

OH

OHCOOH

NH2 COOH

CO2

H+

H+

O2/Me2+

e-

OH

OHOH

NH2

OH

OH2OH2

OH2

CH2

N

N

COOH

CH2

COOH

+.

OH2

CH2 O

OH2

OH2

OH2NH2 COOH

CO2 OH2,

OH2

CH2 O

OH2

OH2

A

C

G F

E

OH

OHOH

NH

OH

OHCOOH

OH

OHOH

NH

O

OHCOOH

OH

OHOH

NH

OH

COOHOH

OH

OH

NH

+

OH

COOHOH

OH

OH

O

O

OH

O CHOOH

OH

OOH OH

CH2OHOH

OOH O

CH3OH

O

OOH OH

CH3

NH2 COOH

NH2 COOH

OH2

B

D

OH2

CH3COOH

HCOOH

(Davidek et al., 2002)


Loss of Glc under boiling conditions:- rapid loss at pH 10-12- high loss at pH 8-12

(Ames, 1998)

Glu

cose

loss

(%)

Lysi

ne lo

ss (%

)

Loss of Lys under boiling conditions:- slower decrease at pH 10-12- small loss at pH 4-9


pH decrease and browning as affected by the type of sugar

Glucose-casein

6,0

6,2

6,4

6,6

6,8

0 10 20 30 40

A420

0

2

4

6

8

pH

browning

pH

Time (min)

Fructose-casein

6,0

6,2

6,4

6,6

6,8

0 10 20 30 40

A420

0

2

4

6

8

pH

pH

browning

Time (min)

Galactose-casein

6,0

6,2

6,4

6,6

6,8

0 10 20 30 40

A420

0

2

4

6

8

pH

browning

pH

Time (min)

(van Boekel et al., 2000)


Colour and flavour formation via Maillard reactions depend very much on the pH

(Hayashi & Namiki, 1986) (Shaw et al, 1990)

Rhamnose + L-prolineGlucose + β-alanine

pH 9.3▲ pH 6.4

pH 3.5


Major reaction pathways as affected by the pH

Glucose + RNH2

Schiff base

1,2-Enaminol

Amadori compound

2,3-Endinol

1-DO

4-DO

1-deoxyosoneroute

4-deoxyosoneroute

C1 + C5C2 + C4C3 + C3

Retro-aldol C2 + C4

Retro-aldolC3 + C3

pH < 5 pH > 7

3-DO 3-deoxyosoneroute

C1 + C5

fragmentation


Major steps of the early stage of the Maillard reaction

CH

CH

O

CH

OH

CH OH

CH OH

CH2OH

OHO

NH R

CH2OH

OHOH

OH

CH

CH

NH

CH

OH

CH OH

CH OH

CH2OH

OH

OH R CH

CH

N

CH

OH

CH OH

CH OH

CH2OH

OH

R

NH2R H2O-

N-GlucosylamineGlucose Schiff base

+CH

CH

NH

CH

OH

CH OH

CH OH

CH2OH

OH

R

H+

-H+

CH

C

NH

CH

OH

CH OH

CH OH

CH2OH

OH

R CH2

C

NH

CH

O

CH OH

CH OH

CH2OH

OH

R

O

NHR

CH2OH

OHOH

OH

Amadori compoundN-Glucosylamine 1,2-Enaminol


1,2-enolisation

CH

C

NH

CH

OH

CH OH

CH OH

CH2OH

OH

R

1,2-Enaminol

CH2

C

NH

C

OH

CH OH

CH OH

CH2OH

OH

R

2,3-enolisation

2,3-Endiol

CH2

C

NH

CH

O

CH OH

CH OH

CH2OH

OH

R

Amadori compound

CH2

C

NH

C

O

CH

CH OH

CH2OH

OH

R CH2

C

NH

C

O

CH2

CH OH

CH2OH

R

O

- H2O

4-Deoxyosone

N+

O

R

OH

Pyridaine derivative

OOH O

5-Hydroxymethylfurfural(HMF)

, HCOOH

O

OOH OH

Pyran-4-one derivative

, CH3COOH

CH

C

NH

CH

OH

CH OH

CH OH

CH2OH

R+

CH

C

O

CH2

O

CH OH

CH OH

CH2OH

- H2O

H2O

- R-NH2

- H+

H+

3-Deoxyosone

CH

CH OH

CH OH

CH2OH

O

CHO

CHO

CH

C

N

CH

O

CH OH

CH OH

CH2OH

OH

R CH

C

CH

O

CH OH

CH OH

CH2OH

OH

O

O2 / Me2+

H2O

- R-NH2

Glucosone

CH2

C

C

OH

CH OH

CH OH

CH2OH

O

CH3

C

C

O

CH OH

CH OH

CH2OH

O

- R-NH21-Deoxyosone


Formation of acetic acid (Phosphate buffer, 0.2 mol/L, 120°C)

1721

38

1.10.50.3

10

1513

2833

38

0

10

20

30

40

50

60

0 60 120 180 240 300Time (min)

Ace

tic a

cid

(mm

ol/L

)

pH 4pH 6pH 8

From glucose/glycine

1.84.54

252928

5053 56

0

10

20

30

40

50

60

0 60 120 180 240 300 360 420 480Time (min)

Ace

tic a

cid

(mm

ol/L

)

pH 4pH 6pH 8

From Amadori compound

Glc Gly Glucosylamine 1,2-enamine Amadori compound

2,3-endiol

3-DO 1-DO

+

(Davidek et al., 2003)


Formation of some C2 and C3 degradation products by the Maillard reaction

CH3OH

OO

R

HOO

OHR CH3

OO

H+1-Deoxyosone

OR

O

H

HO

+

HO O

OHR O

H O

H

OH

Glycosone

pH 9.3∆ pH 6.4

pH 3.5

(Hayashi & Namiki, 1986)

(Glucose/β-alanine)


Role of buffer on pH control and reactivityin the Maillard reaction

(Xyl/β-Ala, pH 7.3)

Mechanism:

- Nucleophilic addition- Proton abstraction from α-position- Enolisation: A, sugar isomerisation- Dehydration: B, 3-deoxyosone formation

Conclusion:

Intramolecular proton abstraction with XO2-

→ more efficient, catalytic effect

Intermolecular proton abstraction with OH-

(Rizzi, 2004)


Formation of the Amadori compound in the Glc/Gly system

OHHO

O

NH

OHHO

COOH

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

0 100 200 300 400

Time (min)

Phosphate buffer

0

1

2

3

4

5

6

0 100 200 300 400

C (m

mol

/L)

Time (min)

No buffer

(D-glucose 0.1 M and glycine 0.1 M, H2O or phosphate buffer 0.1 M, T = 90°C)

pH 5,▲ pH 6; pH 7

(Kervella et al., 2002)


Summary: pH effects on reactions occurring in the Maillard cascade

pH changes depend on:

Formation of acid/base : HCOOH, HOAc, glycolic acidConsumption of acid/base : aminoketones → pyrazines Buffering capacity of Maillard/food system

Typical reactions in the ‘Maillard reaction’:

Amino/carbonyl reactions : basicity of α-NH2, pKa

Aldol/retro-aldol reactions : ‘alkaline’ conditionsEnolisation and elimination : ‘alkaline/acidic’ conditionsRadical reactions : ‘alkaline conditionsOxidation and reduction


Impact aroma compounds with caramel/sweet character: O-Heterocycles

O

OH

O O

OH

O O O

OH

(0.04) (n.d.)(0.02)

(10)O

OHO

O

OHO

O

OHO

O

OHO

(8300)(40)

Odour thresholdsin water (µg/L)

OH

OO

OHO

OH

OO

OHO

(2800) (61000) (17)(44)

(Wild, 1988)


Formation of Furaneol (1 M aq. solution, no buffer, pH= const., T= 90°C)

(Blank et al., 1997)

30From glucose and glycine

0

10

20

0 20 40 60 80 100 120

O

OHO

Time (min)

Fura

neol

(m

g / m

ol)

0

50

100

150

200

0 20 40 60 80 100 120

From Amadori compound

pH 6.0pH 7.0

Time (min)

0

0.2

0.4

0.6

0.8

1.0

1.2

0 20 40 60 80 100 120

Fru-

Gly

(mol

)

0 20 40 60 80 100 1200

0.04

0.08

0.12

0.16

0.20


Formation of furaneol from Amadori compounds via acetylformoine

c

O

OH OH

OH

H OHOOO

O

O OH

- H2O

N

OH

OH

OH

O

OH

OOH

R

H

Amadori compound

a

- Amino acid

O

O

OH

OH

OH

1-Deoxyosone

O

OH O

OH

bO

OH

OH

O- H2O

Acetylformoine



Degradation of pentose sugars via the Maillard reaction

(Monti et al., 1998)

pH 3

pH 4

pH 6

OO

O

OHO

(Xyl/Gly, boiling, 2 h)

Pentose

3-DO1-DO

O

OHO

OO

2-FurfuralNorfuraneol


Formation of furylethylether indicating aging flavour of beer

FEE formation is correlated with- high EtOH content,- darker colour, - lower pH.

FEE(Flavour threshold: 6 µg/L beer)

(Vanderhaegen et al., 2003, 2004)


Impact aroma compounds with roasty/sweet character: N-Heterocycles

NO

NO

NOH

2-Acetyltetrahydropyridine(roasty, 0.06)

2-Acetyl-1-pyrroline(roasty, 0.02)

N

S

OHN

O

S

NO

2-Acetyl-2-thiazoline(roasty, 0.05)

2-Acetylpyrazine(roasty, 0.4)(roasty, 0.06)

(Threshold values in ng/L air)


Formation of impact odorants from proline/sugar mixtures at pH 7

O

OHOH

OH

OH

OH N COOH

H

+

NO

OH OO

OH2

NO

O

H

NO

CO2

O

N

NOH

O

H

O

NH

020406080

100120140160180200

Glc/Pro Fru/Pro MG/Pro

Conc

entra

tion

[mg/

mol

Pro

]

ATHP2-AP

0.14 mol%

0.03mol%0.01 mol%

Reaction conditions:Pro (4 mmol) + ‘sugar’ (2 or 0.1 mmol)Phosphate buffer (pH 7.0, 0.1 mol/L)Reflux, 2 h

[O]

2-APATHP

(Schieberle et al., 1995, 1998, 2000)


Increased yields of roasty odorants: Reaction of secondary degradation products

O

OHOH

OH

OH

OH N COOH

H

+

Reaction conditions:1-Pyrroline (10 µmol) + MG or HP (10 µmol)Phosphate buffer (pH 7.0, 0.5 mol/L)Reflux, 30 min

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.5 5.0 5.5 6.5 7.0 8.0 9.0

pHYi

eld

[mol

%]

2-APATHP

NO

OH OO

HP MG

NOH

O

HN

OO

H

[O]

OH2

NO

CO2

O

NO

NH

2-APATHP

(Schieberle et al., 1995, 1998)


Increased yields of roasty odorants from key intermediates: ATHP from HOP

O

OHOH

OH

OH

OH N COOH

H

+

NO

OH OO

OH2

NO

O

H

NO

CO2

O

N

NOH

O

H

O

NH

HOP

HP

0

510

1520

25

3035

40

3 5 7 9pH

ATH

P yi

eld

[mol

%]

HP

HOP

Reaction conditions:HOP (1 µmol)Phosphate buffer (pH 7.0, 0.5 mol/L)Reflux, 30 min

[O]

2-APATHP

(Schieberle et al., 1995, 1998)


Impact aroma compounds with roasty / savoury character: S-containing odorants

Coffee, meatSulfury, coffee-likeOx= 0.01 µg/L H2O

2-Furfurylthiol (FFT)

OSH

Meat, coffeeSulfury, meatyOx= 0.007 µg/L H2O

2-Methyl-3-furanthiol (MFT)

O

SH

Beer, coffeeSulfury, cattyOx= 0.0003 µg/L H2O

3-Methyl-2-buten-1-thiol

SH

Coffee, beerSulfury, cattyOx= 0.003 µg/L H2O

3-Mercapto-3-methylbutyl formate

SH

O H

O

Potato, beerCooked potato-likeOx= 0.2 µg/L H2O

Methional

S O Cabbage, beerSulfury, cabbage-likeOx= 0.006 µg/L H2O

Dimethyltrisulfide (DMTS)

SS

S


Cysteine and pentose sugars are important precursors for thiols

0

5

10

15

20

25

3 5 7pH

FFT

amou

nt [u

g]

RibGlc

OSH

0

10

20

30

40

50

60

3 5 7pH

MFT

am

ount

[ug]

O

SH

CHO

CH2OH

+

COOH

NH2

SH

OH

OH

OH

O

O OH

O

SHH2S

O CHO OSH

H2S

2-Furfural 2-Furfurylthiol

Norfuraneol 2-Methyl-3-furanthiol

Ribose

Cysteine

(Münch et al., 1997)


Formation of 2-furfurylthiol (FFT) from2-furfural in the presence of H2S

OO

HO

OH

SH

HO

SH

HO

SH+ H2S - H2O H2S

H+ H

+S,

+

ThiosemiacetalFFT

(550 µg, 0.5 mol%)(1 mmol each) H2S(25 µg, 0.02 mol%) Cysteine


(Zheng & Ho, 1994)

First-order kinetics ofH2S release from cysteine

Conditions:0.1 M Cys, buffer, 100 °CDetection:Sulfide/silver ion selectiveelectrode


Formation of 2-furfurylthiol (FFT) from sugar fragments and H2S


0

20

40

60

80

3 5 7pH

FFT

[ug]


MFT FFT

Reaction conditions:

Precursors (each 1 mmol)

Phosphate buffer (50 mL, 0.5 mol/L)

Autoclave (145 °, 20 min)

0

50

100

150

200

250

300

350

3 5 7

pH

Amou

nt [u

g]

FFTMFT

0.3 mol%

Mechanism:

Aldol-type condensation

Cyclisation, dehydration

(Hofmann & Schieberle, 1998)


Formation of S-containing odorants from alcohols under acidic conditions

Reaction conditions:Alcohol, acetate buffer, pH 4.0, 100 °COR

OHOH

OR

ORS

R' SR'

H+ , - H2O

R’S-amino acid

++

Mechanism:Acid-catalysed alkylation of amino acid S-atom via cationic intermediates.Unsaturated alcohols form electrophilicspecies in acidic media reacting with ambient nucleophilic sites.

(R, R’: H, Me)

R

OO

OH

OHO SH

Light SH.

.

(Sakuma et al., 1991)

Sunstruck off-flavour in beer:Light-induced radical reaction of isohumulone and an SH-source

(riboflavin-photosensitized reaction)

(Rizzi, 2000)


Formation of methional and DMTS during accelerated aging of beer

(Gijs et al, 2002)

S O

SS

S

S OH

O

CH3SHCH3SH S

SS

SS + S

SS

Methional DMTS DMSMethanethiol DMDS

Beer pH

Reaction conditions:Storage for 5 days at 40 °C

Mechanism:Strecker degradation of methionine to form methional.Formation of sulfite/aldehyde adducts trapping methional.→ Higher DMTS amounts at lower pHvia disproportionation of DMDS


The role of pH in the Maillard reaction:Conclusions

• Many steps in the Maillard cascade are affected by the pH

• pH effect can be different, favouring reactions under acidic or

alkaline conditions

• Buffer may have various tasks: i.e. constant pH (reaction control),

catalytic effect (increasing reaction rate)

• Neutral pH (6-7) is often the best compromise for flavour formation

• Final amounts depend on formation & degradation, both of them

are influenced by pH and may lead to off-notes

Thanks for your attention


Back-up slides


Formation of sotolon from 4-hydroxy-L-isoleucine (HIL) and its lactone

OHOH NH2

OO

OH

O

HIL Sotolon

O

OO

NH2

O

HIL-Lactone

O

OR

RR

R

Carbonyl compound Sotolon yield (mol%)(from HIL-Lactone)

Methylglyoxal

2,3-Butanedione

35.9

0.3

Sotolon yield (mol%)(from HIL)

7.4

<0.1



Lactonisation of 4-hydroxy-L-isoleucine (HIL) is favoured under acidic conditions

OHOH NH2

O

HIL

O

NH2

O

HIL-Lactone

H +

0

0.1

0.2

0.3

0.4

0.5

0.6

Mol

ar ra

tio H

IL-L

acto

ne /

HIL

pH 7 pH 6 pH 5 pH 4 pH 3

Reaction conditions : 100°C, 1 h, phosphate bufferAnalytical technique : FAB-MS

(Blank et al., 1997) PAGE 10NESTLE RESEARCH CENTER


Formation of sotolon from hydroxyisoleucine(HIL) and its lactone: Influence of the pH

pH

0.0

10.0

20.0

30.0

40.0

50.0

60.0

2.5 3.5 4.5 5.5 6.5 7.5

Yiel

d (m

ol %

)

Methylglyoxal + HIL-LactoneMethylglyoxal + HIL

→ Optimum: pH 5-6


Sotolon formed from 4-hydroxy-L-isoleucineby thermally induced oxidative deamination

OHOH NH2

O

O

OH

O

O

NH2

O

Sotolon

H +

H O2

HO

OH O2

O

N

O

H CH C

O

CH3

O

N

O

CH C

OH

CH3

H O2NH2

OCO2

OH

O

N

OH

O

OH

N

OH

H O2 OH

O

Strecker aldehydeNH2

O

MG



Formation of secondary degradation products: 1-Pyrroline and methylglyoxal

CH3OH

OO

R

HOO

OHR

+CH3

OO

HRARetro-aldol

reaction

Strecker reaction

Documents

The Role of pH in Maillard-Type Reactions 2004-09-08 NRC/FCI - IBk/Louvain Nestlé Research Center Outline of the presentation Basic principles of pH/Maillard • pH versus reactivity