ASSESSING THE GENERATION AND BIOACTIVITY OF NEO-FORMED COMPOUNDS IN THERMALLY TREATED FOODS

ISBN: 978-84-96101-76-0

Cristina Delgado-Andrade José Ángel Rufián-Henares (Editors)

Assessing the Generation and Bioactivity of neoformed Compounds in Thermally treated Foods

atrio editorial

Editorial Atrio

port. cost 2009

1

12/3/09, 13:55

ASSESSING THE GENERATION AND BIOACTIVITY OF NEO-FORMED COMPOUNDS IN THERMALLY TREATED FOODS

Editors:

CRISTINA DELGADO-ANDRADE JOSÉ ÁNGEL RUFIÁN-HENARES

ASSESSING THE GENERATION AND BIOACTIVITY OF NEO-FORMED COMPOUNDS IN THERMALLY TREATED FOODS

GRANADA , 2009

© Los Autores © Editorial Atrio, S.L. EDITORIAL ATRIO, S.L. C./ Dr. Martín Lagos, núm. 2 - 1.º C 18005 Granada Tlf./Fax: 958 26 42 54 e-Mail:atrioeditorial@ telefonica.net ISBN: 978-84-96101-76-0 Depósito Legal: Gr.-000/2009

Summary

1. Non-enzymatic browning: The case of the Maillard reaction JOSÉ ÁNGEL RUFIÁN-HENARES; CRISTINA DELGADO ANDRADE; FRANCISCO J. MORALES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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2. Colour and fluorescence measurement as unspecific markers for the Maillard reaction CRISTINA DELGADO-ANDRADE; JOSÉ ÁNGEL RUFIÁN-HENARES; FRANCISCO J. MORALES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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3. Gastrointestinal digestion as first conditioning of nutrient bioavailability CRISTINA DELGADO-ANDRADE; ANA HARO; ROSA CASTELLANO; JOSÉ ÁNGEL RUFIÁN-HENARES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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4. Antimicrobial activity of Maillard reaction products JOSÉ ÁNGEL RUFIÁN-HENARES; CRISTINA DELGADO-ANDRADE . . . . . . . .

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Non-enzymatic browning: The case of the Maillard reaction JOSÉ ÁNGEL RUFIÁN-HENARES Department of Nutrition and Food Science, Faculty of Pharmacy, University of Granada, Spain. CRISTINA DELGADO-ANDRADE Unit of Animal Nutriton, Estación Experimental del Zaidín, CSIC, Granada, Spain FRANCISCO J. MORALES Instituto del Frío, CSIC, Madrid, Spain

Non-enzymatic browning (NEB) is a set of complex reactions produced in thermally treated foods giving rise to the formation of brown colours (Cheftel and Cheftel, 1980). NEB produces undesirable effects during the processing and storage of different liquid foods such as milk or fruit juices whereas for other solid foods the changes are favourable (in the case of bread, breakfast cereals, candies, coffee, chocolate, etc.). NOB can be divided in three different reactions called ascorbic acid degradation, caramelisation (degradation of sugars) and the Maillard reaction (sugar-amino acid reaction). The conditions where such reactions take place are reviewed in the next diagram.

Mechanism

Oxygen

Amino groups

Optimum ph

Heat

Aw

Ascorbic acid degradation

yes/no

no

slightly acid

mild

medium/high

Caramelisation

no

no

basic/acid

strong

low

Maillard reaction

no

yes

basic

mild

low/medium

1. ASCORBIC ACID DEGRADATION L-ascorbic acid, or vitamin C, is a highly water-soluble and strongly reducing substance with acidic properties. This chemical behaviour is related to its enodiol structure conjugated with a carbonyl group (a lactone) which makes this molecule

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José Ángel Rufián-Henares; Cristina Delgado-Andrade; Francisco J. Morales

very sensitive to different ways of degradation (Finholt y col., 1965). The degradation of ascorbic acid is accomplished in the absence of amino groups, slightly acidic pH, water activity medium/high and moderate temperature. There are two different pathways, one oxidative and another non-oxidative, although one of the main differential characteristics among them is the higher production of furfural in the nonoxidative pathway. The selection of the oxidative or the non-oxidative pathways depends on the presence of metallic catalysts. In an acidic medium, the non-oxidative degradation of ascorbic acid starts with the hydrolysis of the lactone group, followed by decarboxilation and dehydration to diketogulonic acid and then, 3-deoxypentosone and furfural. During the oxidative degradation, the ascorbic acid molecule is transformed to dehydroascorbic acid, which is the main precursor of the degradation products. Then, the dehydroascorbic acid is converted to diketogulonic acid, which is the same molecule obtained in the non-oxidative pathway. The scheme of the reaction is shown in figure 1.

ketonisation

No pa n- ox thw yd ay ativ e

Líneas con trazo más oscuro: principales formas con actividad vitamínica. H2A: ácido + soft rA educing córbico reducido. HA: monoanión del ácido ascórbico. A: ácido dehidroascórbico. : radical

agents

ión ascorbato. DKG: ácido dicetogulónico.

Mn+: catalizador metálico. HO.2: radical

O

Oxydative Oxydative droperoxilo. DP: 23-desoxipentosa. X: xilosa. F: furfural. FA: ácido furancarboxílico. slow

pathway

pathway

(catalysed)

(not catalysed)

b) Degradación enzimática: Es producida gracias a la acción de la enzima ascórbico tiv

e

a idasa y se realiza mediante la transformación del ácido ascórbico en ácido dehidroascórbico, yd

sde donde se producen los correspondientes pigmentos. La enzima

x -o ay necesita on w cobre N ath p

como

factor y es producida principalmente en productos cítricos y sus derivados. Los tratamientos rmicos inhiben la enzima y la falta de oxigeno hace que la acción se produzca a mucha menos locidad, al ser, también, oxígeno-dependiente.

± amino acids reductones

Melanoidins

H2A: reduced ascorbic acid HA: ascorbic acid monoanion. A: dehidroascorbic acid. A+: ascorbate anion. DKG: diketogulonic acid. Mn+: metalic catalyst. HO.2: hydroperoxyl radical. DP: 3-deoxypentose. X: xylose. F: furfural. FA: furancarboxílic acid.

Figure 1 Ascorbic acid degradation pathway

Non-enzymatic browning: The case of the Maillard reaction

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1.2. Caramelisation Caramelisation is another kind of NEB obtained when sugars are heated over their fusion temperature, giving rise to an enol intermediate and final dehydration products (Krow, 1994). This pathway and other relevant reactions can be seen in figure 2. If the reactive sugar is a disaccharide, such as sucrose, first a previous hydrolysis step must be performed in order to release two monosaccharides. Pentoses give rise to the formation of furfural as the main degradation product whereas hexoses produce 5hydroxymethylfurfural (HMF). As an example the formation of HMF can bee seen in figure 3.

Figure 2 Selected sugar degradation reactions

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José Ángel Rufián-Henares; Cristina Delgado-Andrade; Francisco J. Morales

Figure 4. Maillard reaction

Figure 3 1,2 and 2,3-enolisation of D-glucose and formation of HMF, HDF and HAF via 3- and 4-deoxyhexosulose

1.3. Maillard reaction 1.3.1. Chemistry of the Maillard reaction The Maillard reaction (MR) is a set of chain chemical reactions that give rise to the formation of brown pigments with modifications in colour, odor and taste of different thermally treated foods (Hodge, 1958). It usually is produced at lowintermediate water activity and basic pH. The general scheme of the reaction can be seen in figure 4. A) The first step of the MR consists on the condensation of a carbonyl group with an amino one and, after dehydration an unstable Schiff base is formed, which is transformed rapidly in a N-substituted-glycosylamine. This reaction is reversible because of in a strong acidic medium the sugar and the aminoacid can be re-generated. H C = O + N - R H Aldose or aldosa, cetosa ketose

am Amino inoácido prot eina group

OH

(H+ )

C

C = N - R + H 2O NH - R

glicosilam ina o

Glycosylamine m ás generalm ente carbonilam ina

bas e de Schiff, Schiff base inestable

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Non-enzymatic browning: The case of the Maillard reaction

Aldose + RNH2

N-substituted glycosylamine -H2O Amadori rearrangement

1-amino-1-deoxi-2-ketose -2H2O

HMF/Furfural

-2H2O

Reductones

Fission products (carboniles, dicarboniles)

+2H -2H

Dehydroreductone

+NH2 -CO2 Strecker degradation

aldehydes

+NH2

+NH2

Aldols +NH2

+NH2

+NH2

MELANOIDINS (Brown nitrogenous polymers and co-polymers)

Figure 4 Maillard reaction The amino group can be a free aminoacid, the side chain of an aminoacid (like lysine) incorporated in a protein or the amino group of the last aminoacid in each protein. In the case of the carbonyl groups, they are usually reducing sugars, although they can be also carbonyl compounds from the intermediate stages of the MR and lipid oxidation.

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José Ángel Rufián-Henares; Cristina Delgado-Andrade; Francisco J. Morales

B) The next step consists on the irreversible rearrangement of the N-substituted glycosylamine: when the molecule is an N-substituted-aldosylamine, by means of the Amadori rearrangement it is formed the 1-amine-1-deoxy-2-ketose. However, when the starting product is an N-substituted-ketosylamine it is formed a 2-amine-2-deoxy2-ketose by means of the Heyns rearrangement. The Amadori and Heyns products are decomposed, depending on the pH and temperature of the medium, giving rise to the formation of different intermediate compounds: these are the intermediate steps of the MR. C) At low pH a 1,2-enolysation occurred, giving rise to the formation of dicarbonyl compounds (powerful precursors of brown compounds) and finally HMF (figure 5). Contrary, at basic pH a 2,3-enolysation takes place (figure 6), being reductones the final compounds. Such reductones can be dehydrated to form dehydroreductones, which form polymers by reacting with amino groups at advances stages of the MR. D) The Amadori compounds can be splitted to different fission products (dicarbonyl compounds) such as acetal or acetaldehyde.

H

H

H

H

H - C - N -R

H - C - N -R

C =O

C - OH

(H - C - OH) n CH2 - OH

1-amino-1-desoxi-2-cetosa 1-amino-1-deoxy-2-ketose

optimum pH

pH óptimo 5,5 5.5

+

H - C =N - R C - OH

+

H - C - OH

H2O

H

H - C (H - C - OH) n - 1

(H - C - OH) n - 1

CH2 - OH

CH2 - OH

forma 1,2 enólica Unstable 1,2 enol (muy inestable)

regeneración del catalizador aminado

H - C = O H - C = O C =O

+

H - C = O

RNH3

C - OH CH2

H2O

acidic mediomedium ácido

C =O

H - C (H - C - OH) n - 1

CH2 - OH

ompuesto alfa3-deoxyoone (3-desoxi hexosona 3-desoxi hexosulosa)

O

CH HOCH2

(H - C - OH) n - 1

CH H2O

CH2 - OH

H - C - OH compuesto dicarbonilo insaturado (3,4-didesoxi 3-eno hexosona = hexosulosa CH2 - OH insaturada)

Figure 5 1,2-enolysation

O

C H

5-Hidroximetilfulfural 5-HMF

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Non-enzymatic browning: The case of the Maillard reaction H

H

CH3 CH2 - N - R

CH2 - N - R C =O C - OH

C =O

C =O H - C - OH

2,3-enolysation enolización 2,3

H - C - OH H - C - OH CH2 - OH

I 1-amino-21-amino-2deoxi-2-ketose desoxi-2-cetosa

C = OH H - C - OH

H - C - OH

R - NH2

H - C - OH

H - C - OH

CH2 - OH

II 2,3-enol forma 2,3 enólica

CH3 C =O

basic pHbásica catálisis

CH2 - OH

III Unstable compuesto metil-alfa compound dicarbonilo (1-desoxi 2,3-diulosa) (inestable)

enolización 3,4 3,4-enolysation CH3

hydrolytic rupture escisiones hidrolíticas

C =O C - OH

aldehidos y cetonas con olor

H - C - OH C - OH C - OH H - C - OH C - OH

C - C

H - C O

CH2 - OH

IV reductone reductona

C - OH

H - C

C - O

HO - C O

CH

H3C - C CH3

O

CH3

CH2 - OH

V

isomaltol isomaltol

furanone furanona

Figure 6 2,3-enolysation E) The interaction of amino acids with dicarbonyl compounds (dehydroreductones fission products) is known as the Strecker degradation and implies the loss of amino acids in foods. As a result of this degradation pathway, new aldehydes with one carbon atom less (lost as CO2) are formed (figure 7). The next steps (F and G) are known as the advanced steps of the MR, where two different classes of compounds are formed: melanoidins and volatile aromatic compounds. F) The volatile aromatic compounds (low molecular weight) are formed directly from the Amadori compounds and don’t need the mediation of free amino groups. However, a 1% of the total volatile compounds produced are formed by the reaction of 2-deoxyglucose with aminoacids. G) Melanoidins are brown polimeric compounds produced by jeans of the condensation of aminated porducts of the intermediate stages of the MR Duch as Nsubstituted pyrrols, 2-formilpyrrols N-substituted, 2-furaldehyde, etc. Melanoidins have a wide molecular weight and different absorbance spectra with maximums in the ultraviolet (280 nm) and visible (420 nm) ranges.

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José Ángel Rufián-Henares; Cristina Delgado-Andrade; Francisco J. Morales HO

HOOC

- C = O

+

- C = O

- C = O

- C = N - CH - R

-am inoáci do αα-amino acid

α -dicarbonilo α-dicarbonyl

C = O

H2N - CH - R H2 O

CO2

- C - OH

O

- C - N = CH - R

H

NH3

- C - OH

+

R - C

- C - NH2

- C = O - CH - OH nuevos

New comcarbonyl puestos compounds carbonilo

Figure 7 Strecker degradation

1.3.2. Variables in the Maillard reaction — Substrates: The main substrates involved in the MR are the carbonyl groups (mainly from reducing sugar) and the amino groups (mainly from free amino acids and those amino acids with side chain amino groups). The first point to take into account is that although the MR is an isomolecular reaction between a sugar and amino acid, the loss of the first one is higher than of the second one, mainly due to other chemical reactions that taking place at the same time (like caramelisation). Low molecular weight reducing sugars are the most reactive species due to their low steric hindrance. The degree of browning produced by different sugars follows the following range: 1st Pentoses: ribose > xilose > arabinose; 2nd Hexoses: galactose > glucose > fructose; 3rd Disaccharides: maltose lactose. In the case of the amino groups, the e-amino group of lysine is the main responsible of the development of the MR in proteical foodstuffs. In this sense, the reactivity of whey proteins is higher than that of caseins, although they are more reactive than soy proteins and these, more reactive than cereal ones (mainly glutenin and glyadin). — pH: The initial pH of foods and their buffering capacity plays an important role in the type and intensity of the MR. At pH