11 Thermal processing and nutritional quality A. Arnoldi, University of Milan 11.1 Introduction The taming of fire, permitting the thermal processing of vegetable foodstuffs in particular, extended enormously the number of natural products that could be used as foods by humans and gave a tremendous impulse to the extraordinary dif- fusion and development of the human population in almost every region of the world (De Bry, 1994). Foodstuffs can be roughly divided in two classes, those that are or are not edible in their raw form. The most important naturally edible foods are meat and milk, which are heated mainly for eliminating dangerous microorganisms, and some fruits, used by plants to attract animals for diffusing their seeds in the environment. In contrast, many plants protect themselves and especially their seeds and tubers from the consumption of insects and superior animals with several antinutritional components that may be deactivated only by thermal treatments. For this reason cereals, grain legumes, and vegetables, such as potatoes, although they are considered the base of a balanced diet in view of the most up-to-date dietary recommendations, are never consumed raw. With the exception of milk, fruit juices, and some other foods, in which a fresh and natural appearance is required, thermal treatments have also relevant hedonistic consequences, as they confer the desired sensory and texture features to foods. Bread and baked products, or chocolate, coffee, and malt are well known products that are consumed world-wide; here thermal treatments produce the characteristic aroma, taste, and colour (Arnoldi, 2001). Such sensory charac- teristics have positive psychological effects that facilitate digestion and therefore contribute to an individual’s well-being. During thermal treatment many reactions take place at a molecular level: ? Denaturation of proteins, with the important consequence of the deactivation of enzymes that destabilise foods or decrease their digestibility, such as lipases, lipoxygenases, hydrolases, and trypsin inhibitors. ? Lipid autoxidation. ? Transformations of minor compounds, for example vitamins. ? Reactions involving free or protein-bound amino acids. The last reactions belong essentially to four categories: ? breaking and/or recombination of intramolecular or intermolecular disulfide bridges; ? reactions of the basic and acidic side chains of amino acids to give isopep- tides (for example Lys + Asp); ? reactions involving the side chains of amino acids and reducing sugars in a very complex process generally named as ‘Maillard reaction’ (MR); ? reactions involving the side chains of amino acids through leaving group elimination to give reactive dehydro intermediates, which can produce cross-linked amino acids. The Maillard reaction is described in this chapter and some information given on those reactions involving the side chains of amino acids. The Maillard reaction, or non-enzymatic browning, is one of the most important processes involving on one hand amino acids, peptides and proteins, and on the other reducing sugars (Ledl and Schleicher, 1990; Friedman, 1996). The MR is a complex mixture of competitive organic reactions, such as tautomerisations, eliminations, aldol con- densations, retroaldol fragmentations, oxidations and reductions. Their interpre- tation and control is difficult because they occur simultaneously and give rise to many reactive intermediates. Soon after the discovery of the MR it became clear that it influences the nutritive value of foods. The loss in nutritional quality and, potentially, in safety is attributed to the destruction of essential amino acids, interaction with metal ions, decrease in digestibility, inhibition of enzymes, deactivation of vitamins and formation of anti-nutritional or toxic compounds. However, while the reaction has its negative effects, the positive effects are considerably greater. 11.2 The Maillard reaction About 90 years ago Maillard (1912) observed a rapid browning and CO 2 devel- opment while reacting amino acids and sugars: he had discovered a new reaction that became known as the ‘Maillard reaction’ or non-enzymatic browning. Nine- teen years later Amadori (1931) detected the formation of rearranged stable products from aldoses and amino acids that became known as the Amadori rearrangement products (ARPs). The development of industrial food processing, especially after World War II, gave a large impulse to research in this field and 266 The nutrition handbook for food processors after some years Hodge (1953) was able to propose an overall picture of the reac- tions of non-enzymatic browning in a review that, after almost 50 years, remains one of the most cited in food chemistry. The mechanism of non-enzymatic browning is generally studied in simple model systems in order to control all the parameters and the results are extrapo- lated to foods quite efficiently. The reactants include reducing sugars. Pentoses, such as ribose, arabinose or xylose are very effective in non-enzymatic browning, hexoses, such as glucose or fructose, are less reactive, and reducing disaccharides, such as maltose or lactose, react rather slowly. Sucrose as well as bound sugars (for example glycoproteins, glycolipids, and flavonoids) may give reducing sugars through hydrolysis, induced by heating or very often by yeast fermentation, as in cocoa bean preparation before roasting or dough leavening. The other reactants are proteins or free amino acids; these may already be present in the raw material or they may be produced by fermentation. In some cases (e.g. cheese) biogenic amines can react as amino compounds. Small amounts of ammonia may be produced from amino acids during the Maillard reaction or large amounts added for the preparation of a particular kind of caramel colouring. A very simplified general picture of the MR may be found in Fig. 11.1. Fol- lowing the classical interpretation by Hodge (1953), the initial step is the con- densation of the carbonyl group of an aldose with an amino group to give an unstable glycosylamine 1 which undergoes a reversible rearrangement to the ARP (Amadori, 1931), i.e. a 1-amino-1-deoxy-2-ketose 2 (Fig. 11.2). Fructose reacts in a similar way to give the corresponding rearranged product, 2-amino-2-deoxy- Thermal processing and nutritional quality 267 Early stage First interactions between sugars and amino groups, rearrangements Fissions, cyclisations, dehydrations, condensations, oligomerisations Polymerisations Intermediate stage Advanced stage Fig. 11.1 Simplified scheme of the Maillard reaction. 2-aldose 3 (Fig. 11.3, Heyns, 1962). The formation of these compounds, that have been separated from model systems as well as from foods, takes place easily even at room temperature and is very well documented also in physiological condi- tions. Here long-lived body proteins and enzymes can be modified by reducing sugars such as glucose through the formation of ARPs (a process known as gly- cation) with subsequent impairment of many physiological functions. This takes place especially in diabetic patients and during aging (Baynes, 2000; Furth, 1997; James and Crabbe 1998; Singh et al, 2001; Sullivan, 1996). A detailed descrip- tion of the synthetic procedures, physico-chemical characterisation, properties and reactivity of the ARPs may be found in an excellent review by Yaylayan and Huyggues-Despointes (1994). Where the water content is low and pH values are in the range 3–6, ARPs are considered the main precursors of reactive intermediates in model systems. 268 The nutrition handbook for food processors HO O H H HO H HO OH H H OH HC OHH HHO OHH OHH CH 2 OH NR HC OH HHO OHH OHH CH 2 OH NHR H 2 C O HHO OHH OHH CH 2 OH NHR O H HO H HO H H OH H NHR OH O OH H H HO H OH OH H H NHR RNH 2 1-amino-1-desoxyaldose 1 1-amino-1-desoxyketose 2 Amadori rearranged product protein or amino acid Fig. 11.2 Mechanism of the Amadori rearrangement. Below pH 3 and above pH 8 or at temperatures above 130°C (caramelisation), sugars will degrade in the absence of amines (Ledl and Schleicher, 1990). Ring opening followed by 1, 2 or 2, 3-enolisation are crucial steps in ARP transfor- mation and are followed by dehydration and fragmentation with the formation of many very reactive dicarbonyl fragments. This complex of reactions is con- sidered the intermediate stage of the MR. Maillard observed also the production of CO 2 , which is explained by a process named the Strecker degradation (Fig. 11.4). The mechanism involves the reac- tion of an amino acid with an a-dicarbonyl compound to produce an azovinylo- gous b-ketoacid 4, that undergoes decarboxylation. In this way amino acids are converted to aldehydes containing one less carbon atom per molecule. These are very reactive and often have very peculiar sensory properties. The aldehydes that derive from cysteine and methionine degrade further to give hydrogen sulfide, 2- methylthio-propanal, and methanethiol: that means that the Strecker degradation is responsible for the incorporation of sulfur in some Maillard reaction products (MRPs). Another important consequence of the Strecker reaction is the incorpo- ration of nitrogen in very reactive fragments deriving from sugars, such as 5. Thermal processing and nutritional quality 269 HO O H H HO H H NHR H OH OH 2-amino-2-desoxy-D-glucose 3 Heyns rearranged product Fig. 11.3 Heyns products. C C O O H 2 N HC COOH R C C O N HC COOH R C C OH N CH R C C OH NH 2 O CH R NH 3 C HC OH O + + 4 Strecker aldehyde 5 Fig. 11.4 Mechanism of the Strecker degradation of amino acids. However, in the last two decades other mechanisms have been proposed. For example, starting from the experimental observation of free radical formation at the start of the MR, Hayashi and Namiki (1981; 1986) proposed a reducing sugar degradation pathway that produces glycolaldehyde alkylimines without passing through the formation of ARPs (Fig. 11.5). Very recently, on the basis of extensive experiments with 13 C- and 2 H-labelled sugars, a detailed reaction scheme was proposed by Tressel et al (1995 and 1998a): the formation of various C 6 -, C 5 -, and C 4 -pyrroles and furans from both intact and fragmented hexoses and amines could be unambiguously attributed to distinct reaction pathways via the intermediates A–C without involving the 270 The nutrition handbook for food processors CHO CHOH CHOH CHOH R' C=N-R CHOH CHOH CHOH R' CH-NH-R CHOH CH=O CHOH R' + RNH 2 - H 2 O reverse aldol reaction 5 Fig. 11.5 Possible pathway for the formation of glycolaldehyde alkylimines proposed by Namiki and Hayashi (1986). CHO OH HO OH OH CH 2 OH OH HO OH OH CH 2 OH NR O H OH OH CH 2 OH NR O H OH CH 2 OH NR O H O OH CH 2 OH NHR O H OH OH CH 2 OH O H OH CH 2 OH HO OH OH CH 2 OH O H CH 2 OH NR OH H O NR O H O CH 2 OH NHR OH H O O NHR N R OH O OH O N R O O O N R polymers polymers 2-deoxypentoses tetroses pentoses C6-pyrroles C6-furans cyclisation cyclisation beta-dicarbonyl route 3,4-dideoxy aldoketose route 3,4-dideoxy aldoketose route 3,4-dideoxy aldoketose route beta-dicar- bonyl route F(c 1 + c 5 ) F(c 2 + c 4 ) F(c 5 + c 1 ) F(c 5 + c 1 ) R-NH 2 3-deoxy aldoketose route (NR) A B C H H H H (NR) Fig. 11.6 Transformation of hexoses and pentoses to C 5 - and C 4 -pyrroles and -furans. (Reproduced with permission from Tressel et al, 1998a) Amadori rearrangement (Fig. 11.6). These pyrroles and furans polymerise very easily to highly coloured compounds that may be involved in the formation of melanoidins. By means of experiments showing that sugars and most amino acids also undergo independent degradation (Yaylayan and Keyhani, 1996), a new concep- tual approach to the MR has been proposed recently by Yaylayan (1997). He sug- gested that in order to understand the MR better, it is more useful to define a sugar fragmentation pool {S}, an amino acid fragmentation pool {A}, and an interaction fragmentation pool {D}, deriving from the Amadori and Heyns com- pounds (Table 11.1). Together they constitute a primary fragmentation pool of building blocks that react to give a secondary pool of interaction intermediates and eventually a very complex final pool of stable end-products. However, most foods contain also lipids that can degrade by autoxidation (Grosch, 1987) giving reactive intermediates, mainly saturated or unsaturated aldehydes or ketones and also glyoxal and methylglyoxal (in common with the Thermal processing and nutritional quality 271 Table 11.1 Composition of the primary fragmentation pools Type of pool Constituents Amino acid Amines fragmentation pool Carboxylic acids {A} Alkanes and aromatics Aldehydes Amino acid specific side chain fragments: H 2 S (Cys), CH 3 SH (Met), styrene (Phe) Sugar fragmentation C1 fragments: formaldehyde, formic acid pool {S} C2 fragments: glyoxal, glycoladehyde, acetic acid C3 fragments: glyceraldehyde, methylglyoxal, hydroxyacetone, dihydroxyacetone, etc. C4 fragments: tetroses, 2, 3-butanedione, 1-hydroxy-2-butanone, 2-hydroxybutanal, etc. C5 fragments: pentoses, pentuloses, deoxy derivatives, furanones, furans C6 fragments: pyranones, furans, glucosones, deoxyglucosones Amadori and Heyns C3-ARP/HRP derivatives: glyceraldehyde-ARP, amino fragmentation pool acid-propanone, amino acid-propanal, etc. {D} C4-ARP/HRP derivatives: amino acid-tetradiuloses, amino acid-butanones C5-ARP/HRP derivatives: amino acid-pentadiuloses C6-ARP/HRP derivatives: amino acid-hexadiuloses, pyrylium betaines Lipid fragmentation Propanal, pentanal, hexanal, octanal, nonanal pool {L} 2-Oxoaldehydes (C6–9) C2 fragments: glyoxal C3 fragments: CHOCH 2 CHO, methylglyoxal Formic acid, acids Maillard reaction) and malondialdehyde (Table 11.1). These belong to a fourth pool, the lipid fragmentation pool {L} (D’Agostina et al, 1998) and in this way the scheme proposed by Yaylayan (1997) was revised to include it (Fig. 11.7). Clear interconnections between the MR and lipid autoxidation have been exten- sively studied in the case of food aromas, where many end-products deriving from lipids and amino acids or sugars are very well documented (Whitfield, 1992), but certainly they may be relevant also for other sensory aspects, such as colour or taste, or for nutrition, although these research areas have been almost completely neglected until the present. Depending on food composition and heating intensity applied, thousands of different end products may be formed in the advanced stage of the MR: they are classified here according to their functions in foods (Fig. 11.8). Very volatile com- pounds, such as pyrazines, pyridines, furans, thiophenes, thiazoles, thiazolines, and dithiazines are of interest, when considering aroma; some low molecular weight compounds relate to taste (Frank et al, 2001; Ottiger et al, 2001), others behave as antioxidants and a few are mutagenic. Polymers (melanoidins) that in sugar/amino acid model systems and some foods such as coffee, roasted malt, or chocolate are the major MRPs, and determine the colour of the food. This review will discuss only the mechanism of formation of MRPs that have some nutritional significance or may be used as molecular markers for quantify- ing the MR in foods. A very detailed description of the pathways leading to most Maillard reaction products may be found in an excellent review by Ledl and Schleicher (1990). 272 The nutrition handbook for food processors {A} {S} {D} {L} polymers Interaction pool Primary fragmentation pool oligomers dimers heterocycles Fig. 11.7 Conceptual representation of the Maillard reaction: generalogy of primary fragmentation pools, interaction pools (containing self-interaction pools as well as mix- interaction pools) and end-products. 11.3 Nutritional consequences and molecular markers of the Maillard reaction in food As the MR involves some of the most important food nutrients, its nutritional consequences must be carefully considered. Researcher attention has previously been focused mainly on milk and milk products, where thermal treatments are necessary for obtaining microbial stabilisation and the preservation of high nutritional quality. Vegetable products, which become edible only after thermal treatments, have been relatively neglected so far. The degradation of sugars per se is never considered a problem because it is only rarely they are lacking in diet. However, free or protein-bound essential amino acids may be damaged irreversibly; the amount of free amino acids in food is always very low and they are important as constituents of proteins. This means that the most relevant nutritional effect of the Maillard reaction is non-enzymatic glycosylation of proteins which involves mostly lysine, whose bioavailability may be drastically impaired. This should be distinguished very clearly from enzy- matic glycosylation, a normal step in the biosynthesis of glycoproteins, in which oligosaccharides are bound to serine or asparagine through a glycosidic bond. The first glycation products are then converted to the Amadori product, fruc- tosyllysine, that eventually can cross-link with other amino groups intramolecu- larly or intermolecularly. The resulting polymeric aggregates are called advanced glycation end products (AGEs). Lysine availability is an important nutritional parameter especially in foods for particular classes of consumers, such as infant formulas (Ferrer et al, 2000). Statistically significant losses of available lysine (about 20%) with respect to raw milk have been reported as a consequence of the thermal treatment applied in the preparation of these foods. Because the reactions of lysine are so relevant in nutrition, over a period of time different MRPs have been proposed as markers of protein glycosylation. Thermal processing and nutritional quality 273 Volatile compounds Maillard reaction Antioxidants Metal chelating agents Toxic compounds Tasty compounds Brown compounds Fig. 11.8 Functional classification of Maillard reaction products. Fructosyllysine is unstable in the acid conditions of protein hydrolysis, produc- ing about 30% furosine, pyridosine (a minor cyclisation product) and about 50% lysine (Fig. 11.9). Furosine was first detected in foods by Erbersdobler and Zucker (1966) and can be easily analysed by HPLC: thus furosine quantification is con- sidered a good estimate of nutritionally unavailable lysine. Milk proteins, owing to their nutritional relevance, have been considered with particular attention. Owing to the presence of lactose, the Amadori compound in this case is lactulo- syllysine and furosine is again a useful marker of lysine unavailability. For this reason several authors have used the furosine method for determining the progress of the Maillard reaction in different foods (Chiang, 1983; Hartkopf and Erbersdobler, 1993 and 1994, Henle et al, 1995; Resmini et al, 1990). However, today very powerful analytical techniques are disclosing new possibilities, permitting, for example, the direct determination of fructosyllysine (Vinale et al, 1999) by the use of a stable isotope dilution assay performed in liquid chromatography – mass spectrometry (LC–MS). This method overcomes the problems of hydrolytic instability of the analyte and the incompleteness of the enzymatic digestion technique. Other possible markers of lysine transformation are N-e-carboxymethyllysine (CML) and 5-hydroxymethylfurfural (HMF) (Fig. 11.10). CML was detected for the first time in milk by Büser and Erbersdobler (1986) and an oxidative mech- anism was proposed for its formation (Ahmed et al, 1986). The formation of HMF in foods has been explained in two ways: via the Amadori products through eno- lisation (in the presence of amino groups) and via lactose isomerisation and degra- dation, known as the Lobry de Bruyn-Alberda van Ekenstein transformation (Ames, 1992). Because of this, it has recently been proposed to measure sepa- rately the HMF formed only by the acidic degradation of Amadori products and 274 The nutrition handbook for food processors CH 2 O HHO OHH OHH CH 2 OH NH (CH 2 ) 4 H C NHRR'OC NH 2 (CH 2 ) 4 H CNH 2 HOOC O H 2 C N H (CH 2 ) 4 O COOH NH 2 NH 3 C (CH 2 ) 4 O NH 2 COOH lysine furosine pyridosine Fig. 11.9 Compounds derived from fructosyllysine decomposition. directly related to the MR, called bound HMF, and total HMF that also derives from the degradation of other precursors (Morales et al, 1997). This method is considered more reliable than the previous spectrophotometric one of Keeney and Bassette (1959). Many authors have observed that the values of furosine, carboxymethyllysine, and HMF are very well correlated (Corzo et al, 1994; O’Brien, 1995). Hewedy et al (1991), however, comparing several damage indicators for the classification of UHT milk have shown that carboxymethyllysine is suitable only for monitor- ing very severe damage, because it is formed only in very small amounts, whereas furosine and HMF have a more general applicability. e-Pyrrolelysine (known also as pyrraline, Fig. 11.10) is another substance that has been proposed to measure the MR in foods. It was observed for the first time in the reaction between glucose and lysine (Nakayama et al, 1980) and is particularly useful in dry foods because it is very stable: for example Resmini and Pellegrino (1994) have proposed a methodology for measuring protein-bound pyrraline in dried pasta. The forma- tion of this MRP parallels very well the formation of furosine. Another useful substance for assessing protein damage is lysinoalanine (see section 11.7). 11.4 Melanoidins The modifications of amino acids described in the preceding section take place also during either mild treatments or short duration treatments at high tempera- Thermal processing and nutritional quality 275 N HOH 2 C CHO (CH 2 ) 4 CO 2 H NH 2 (CH 2 ) 4 CO 2 H NH 2 NH CH 2 COOH N N N H HN (CH 2 ) 4 (H 2 C) 4 NH 2 HO 2 C HO 2 CNH 2 pyrraline carboxymethyllysine pentosidine + Fig. 11.10 Some possible markers of the Maillard reaction. tures, where the changing of food appearance is hardly perceptible. However, many processes usually dealing with the processing of vegetables, such as bread baking, roasting of coffee and nuts, and kiln drying of malt as well as roasting of meat require severe thermal treatments. In these cases the Maillard reaction is responsible for colour formation, a very critical parameter in determining food quality. The most important contribution to colour comes from polymers, known as melanoidins that in some foods are the major MRPs. Their structure is still elusive but many methodological difficulties have slowed down the progress of knowledge in this field. Some attempts have been made to isolate melanoidins from such foods as soy sauce (Lee et al, 1987), dark beer (Kuntcheva and Obretenov, 1996), malt or roasted barley (Milic et al, 1975; Obretenov et al, 1991), coffee (Maier and Buttle, 1973; Steinhart and Packert, 1993; Nunes and Coimbra, 2001), but their very complex and probably non-repetitive structure has limited structural characterisation. Studies on model systems have clearly indicated that reducing carbohydrates and compounds possessing a free amino group, such as amino acids, either in free or protein-bound form, are the basic material for their formation (Ledl and Scleicher, 1990). They are reported to have molecular weight up to 100 000 Da, and to possess structural features, elemental analysis and degrees of unsaturation depending on the reaction conditions. As already indicated at the beginning of this review, the Maillard reaction is often studied through model systems; however, in the case of melanoidins this approach shows limitations. Many authors have used model systems to isolate and characterise low molecular weight coloured compounds (Rizzi, 1997; Arnoldi et al, 1997; Ravagli et al, 1999; Tressl et al, 1998a and 1998b; Wondrack et al, 1997). Currently the most active in this field are Hofmann (Hofmann, 1998a, b, c, d) and co-workers (Hofmann et al, 1999). However, it has been clearly demon- strated that completely different compounds are produced by reacting glucose with single amino acids or b-casein (Hofmann, 1998c). With amino acids the majority of coloured compounds have molecular weights below 1000 amu, whereas the reaction between glucose and casein gives rise to products with much higher molecular weights (Hofmann, 1998a). Moreover, only very rarely has it been demonstrated that the chromophores isolated from amino acids/sugars model systems may be incorporated in melanoidins, as in the case of the free radical chromophore named CROSSPY (Fig. 11.11), which was successfully identified in several processed foods, such as coffee and bread crusts by EPR (Hofmann et al, 1999). However, the formation of melanoidins in real foods does not involve simply proteins and sugars: biopolymers (proteins in animal proteins, both proteins and polysaccharides in vegetables) probably act as a non-coloured skeleton bearing a variety of chromophoric substructures. Thus the melanoidins separated from coffee brews contain about 33% polysaccharides, 9% proteins, and 33% polyphe- nols (Nunes and Coimbra, 2001). The polyphenol substructures derive from chlorogenic acids (Heinrich and Baltes, 1987) that disappear during coffee roasting. Physico-chemical properties of melanoidins other than colour may be 276 The nutrition handbook for food processors important in foods. For example, in espresso coffee brew, they may have foaming properties (Petracco, 1999) that stabilise the foamy layer on top of the beverage, which is well known by the Italian term crema. Beside their obvious importance in determining the brown colour of roasted foods, in recent years great interest has developed around melanoidins for their possible role in physiology and in food stabilisation: in fact some authors (Anese et al, 1999; Nicoli et al, 1997) have demonstrated that they possess antioxidative activity. Antioxidants are able to delay or prevent oxidation processes, typically involving lipids, which greatly affect the shelf-life of foods. In coffee, the antiox- idant activity (attributed to the development of Maillard reaction products) increases with roasting up to the medium-dark roasted stage, then decreases with further roasting (Nicoli et al, 1997), possibly indicating a partial decomposition in the antioxidative compounds. 11.5 Transformations not involving sugars: cross-linked amino acids Another kind of transformation of the side chains of protein-bound amino acids induced by thermal treatments of foods, particularly in basic conditions, is the formation of cross-linked amino acids. A dehydroalanine residue may be formed through elimination of a leaving group from protein-bound serine, O- phosphorylserine, O-glycosylserine, or cystine, and may undergo Michael addition by another nucleophilic amino acid residue (Fig. 11.12). For example the e-amino group of lysine may react to give a secondary amine, which is nor- mally indicated with the trivial name of lysinoalanine (LAL) (Maga, 1984). Anal- ogous reactions may involve ornithine to give ornithinoalanine (OAL), cysteine to give lanthionine (LAN), and histidine to produce histidinoalanine (HAL) Thermal processing and nutritional quality 277 N N protein protein Fig. 11.11 Structure of the radical cation CROSSPY. (Finley and Friedman, 1977). Both nitrogens of histidine may react, giving rise to the regioisomers N p -HAL and N t -HAL (Fig. 11.13) (Henle et al, 1993). The formation of cross-linked amino acids does not involve the participation of reducing sugars and is particularly extensive when proteins are submitted to aqueous alkali treatments. Such treatments include those used in the preparation of soy protein concentrates and in the recovery of proteins from cereal grains, milling by-products, and oilseeds, such as cottonseeds, peanuts, safflower seeds and flaxseeds, and in the separation of sodium caseinate. Other alkali procedures are commonly used for destroying microorganisms, preparing peeled fruits, and inducing fiber-forming properties in textured soybean proteins, used, for example, in the preparation of meat substitutes. A recent review lists the processes and foods that have been studied for the formation of cross-linked amino acids (Friedman, 1999). The main feature of these compounds is that they are stable during acidic protein hydrolysis and are relatively easy to analyse when other 278 The nutrition handbook for food processors H 2 CC H Protein NH O ProteinZ H 2 C C Protein HN O Protein OH elimination Z = OPO 3 H 2 , phosphoserine = O-glycoside, glycoserine = S-S-CH 2 CH(NH 2 )COOH, cystine lysinoalanine dehydroalanine protein lysine Fig. 11.12 Mechanism of the formation of dehydroalanine residues in protein chains. HN CH 2 (CH 2 ) 3 CH 2 CH * COOH NH 2 CH * COOH NH 2 HN CH 2 (CH 2 ) 2 CH 2 CH * COOH NH 2 CH * COOH NH 2 N N H 2 C CH 2 CH * CH * NH 2 COOH NH 2 COOH LAL OAL N-pi-HAL N N CH 2 CH 2 CH * NH 2 COOH N-tau-HAL C H H 2 N COOH Fig. 11.13 Main cross-linker amino acids. compounds that derive from modification of the amino acid side chains in pro- teins, such as isopeptides, must be considered. This makes them useful in quality control as molecular markers of the processes applied in food preparation (Pel- legrino et al, 1996). LAL is certainly the most frequently studied cross-linked amino acid. Many investigations have been devoted to investigating the effects of its presence on protein functionality and nutritional value, because LAL acts as a bridge or a cross-linker between two different parts of the protein chain (Pellegrino et al, 1998). It can thus impair the approach of the enzymes and consequently decrease protein digestibility (Anantharaman and Finot, 1993; Savoie et al, 1991). The nutritional consequences of LAL formation in foods have been extensively reviewed (Karayiannis et al, 1980; Maga, 1984; Friedman, 1999), adverse effects on growth, protein digestibility, protein quality, and mineral bioavailability and utilisation were observed (Sarwar et al, 1999). There are also some concerns about toxicity; LAL has been shown to provoke lesions in rat kidney cells causing nephrocitomegaly (Friedman et al, 1984; Friedman and Pearce, 1989). Although these effects seem very species specific, such observations promoted investigation on humans, in particular on preterm infants (Langhendries et al, 1992). The higher level of Maillard reaction prod- ucts and LAL in infant formulas compared to breast milk had no influence on creatinine clearance or electrolyte excretion and there was no evidence of tubular damage as determined by the urinary excretion of four kidney-derived enzymes. Feeding with formulas, however, did result in a general increase in urinary micro- protein levels. Some authors have investigated the presence of LAL in infant formulas in the 1980s (Fritsch and Klostermeyer, 1981; de Koning and van Rooijen, 1982; Bellomonte et al, 1987): the data were in the range of 150–2120mg/g protein. Some dried and liquid samples have been analysed very recently by us (D’Agostina et al, 2002): the LAL contents in dried formulas were negligible, whereas in liquid samples they were lower than 80mg/g protein in adapted for- mulas, 80–370mg/g protein in follow-on formulas, and about 500mg/g protein in growing milk, indicating that current products are much better than older ones and that producers have made considerable efforts to improve the manufacturing procedures especially in adapted formulas. In particular, the replacement of in- bottle sterilisation by UHT treatments can reduce the thermal damage in liquid samples (Rennen and Vetter, 1988). LAL contents ranging from 150 to 800mg/g protein were observed in liquid samples for enteral nutrition, which are mainly based on casein, a protein more sensitive than lactalbumin to this reaction (Boschin et al, 2002). Some studies were focused also on histidinoalanine, that was detected for the first time by Finley and Friedman (1977) in soybean protein isolates treated with alkali. The HAL content of several proteins (bovine serum albumin, bovine tendon collagen, and casein) heated in neutral pH buffer was greater than their LAL content (Fujimoto, 1984). This probably derives from the lower pKa of the NH group of histidine (pK a = 5.5) in respect to e-NH 3 + of lysine (pK a = 10). Thermal processing and nutritional quality 279 Analysis of various milk-protein containing foods, such as heated skim milk, ster- ilised milk, and baby formulas, permitted detection of amounts of HAL between 50 and 1800mg/g protein, comparable to LAL amounts (Henle et al, 1993; Henle et al, 1996). However, no toxicological effects have been reported for HAL. 11.6 Metabolic transit and in vivo effects of Maillard reaction products These topics are considered in detail in an excellent review by Faist and Erbersdobler (2001) that has been used extensively to prepare this section. The two authors suggest dividing MRPs into three classes: melanoidin precur- sors (reactive low molecular weight compounds), premelanoidins (non-polymeric end products), and melanoidins. At the outset, it is important to underline that most data have been obtained by feeding experiments in rats, whereas only the Amadori compound fructosyllysine has been administered to humans (Erbersdobler et al, 1986; Lee and Erbersdobler, 1994). 11.6.1 Absorption The intestinal absorption of the Amadori compounds occurs by passive diffusion (Faist and Erbersdobler, 2001). Amounts varying from 60 to 80% of ingested fructosyllysine is excreted in the urine, whereas only 1–3% undergoes faecal excretion (Faist and Erbersdobler, 2001). In humans trials (Erbersdobler et al, 1986; Lee and Erbersdobler, 1994) about 3% of orally administrated casein- bound fructosyllysine is excreted in urine, and only 1% via the faeces. Higher transit rates have been reported for infants (Niederweiser et al, 1975), as 16 and 55% of casein-bound fructosyllysine ingested from glucose-containing formula were excreted in the urine and faeces, respectively. The fate of most protein- bound fructosyllysine in humans remains completely obscure, indicating that its fate is probably metabolisation, degradation by intestinal microorganisms, or accumulation in different tissues. Microbiological degradation up to 80% has been demonstrated by Erbersdobler et al (1970). Consistent amounts of CML are formed in foods containing milk protein which are severely treated and very small amounts of CML, detectable in the urine of human infants, are now considered normal constituents of urine (Wadman et al, 1975). Among the few other premelanoidins that have been inves- tigated, furans and pyrroles are known to inhibit intestinal carboxypeptidases and aminopeptidases (?ste et al, 1986). HMF, by radio-labelling experiments, has been demonstrated to accumulate mainly in the kidney and less in the bladder and liver (Germond, 1987). In conclusion, taking into account the data collected to date, it can be said that three different mechanisms are likely to be involved in the metabolic transit of early and advanced MRPs: (1) intestinal degradation by digestive or microbial enzymes and subsequent adsorption of the MRPs or their degradation products; 280 The nutrition handbook for food processors (2) metabolisation of MRPs themselves or their degradation products, probably neither acting as metabolically inert substance; (3) different retention mechanisms in various tissues and organs (Faist and Erbersdobler, 2001). In the case of melanodins, studies indicate that they are partially absorbed in the intestine; the level is low for high molecular weight fractions, and high for low molecular weight fractions. Specific transport mechanisms are still unknown. It is speculated that the fractions absorbed are not used by the organism and are excreted slightly modified or unmodified with the urine. Kidneys retain these compounds more than do other organs, such as liver. For the nonabsorbable low molecular weight fractions, intestinal degradation by digestive or microbial enzymes may be postulated while the high molecular weight fraction is not degraded (O’Brien and Morrissey, 1989). 11.6.2 Antioxidant activity In vivo effects of MRPs and melanoidins may be classified as primary, attributed to specific actions, and secondary, based on interaction with other nutrients (Faist and Erbersdobler, 2001). Most of the secondary nutritional effects may be cor- rected with a suitable dietary supplementation. An important primary effect of browning is the formation of antioxidants, compounds that are able to delay or prevent oxidation processes, typically involv- ing lipids. Such antioxidants greatly affect the shelf-life of foods but may also benefit health (Halliwel, 1996), especially in the prevention of cancer (Kim and Mason, 1996), cardiovascular disease (Maxwell and Lip, 1997) and ageing (Deschamps et al, 2001). The formation of antioxidants in browning has been observed in several different systems, for example sugar/amino acids model systems (Lignert and Eriksson, 1981), model melanoidins (Hayase et al, 1990), and honey/lysine model systems (Antony et al, 2000). They have also been seen in heated or roasted foods, such as coffee brews (Nicoli et al, 1997). However, the processing con- ditions should be chosen very carefully: in coffee, for example, the antioxidant activity increases with roasting up to the medium-dark roasted stage, then decreases with further roasting (Nicoli et al, 1997). This experimental observa- tion is explained by the partial decomposition of the antioxidant compounds. 11.6.3 Activation of xenobiotic enzymes Much interest has been raised also by the possible activation of xenobiotic enzymes by MRPs. Induction of detoxifying enzymes, either by natural or synthetic substances, is still a promising chemopreventive strategy (Faist and Erbersdobler, 2001). Naturally occurring substances in foods have been shown to serve as antimutagens, which may function as chemical inactivators, enzymatic inducers, scavengers and antioxidants. The modulators can act through enzyme systems by inducing phase-I and phase-II enzymes or by altering the balance of different enzyme activities. Thermal processing and nutritional quality 281 Phase-I metabolic transformations include reduction, oxidation and hydrolytic reactions in order to release or induce functional or reactive groups from or into the xenobiotic substances. Phase-II transformations are mostly conjugation reac- tions of the parent xenobiotics, or phase-I metabolites, with sulphur-containing amino acids or glutathione. The conjugation reactions facilitate transport and hence elimination. Thus the balance between phase-I and phase-II enzymes is very critical (Prochaska and Talalay, 1988). The most potent inducers of these enzymes in foods are phenolic compounds and antioxidants. Antimutagenic prop- erties of MRPs have been noted by Kim et al (1986) and are attributed to the inhibition of mutagenic activation through enhanced detoxification of reactive intermediates (Kitts et al, 1993; Pintauro and Lucchina, 1987). Because several MPRs or melanoidins exhibit antioxidant properties, they may inhibit phase-I mutagenic activating enzymes and may induce detoxifying phase-II enzymes. CML-casein increases significantly the activity of phase-II glutathione-S- transferase in kidney isolates, whereas LAL-casein has no effect (Faist et al, 1998; Wenzel et al, 2000). 11.6.4 Other activities Melanodins possess the ability to bind metals, such as copper and zinc (O’Brien and Morrissey, 1989; Andrieux et al, 1980 and 1984; Furniss et al, 1986). Some antibiotic activity against both pathogenic or spoilage organisms, includ- ing Lactobacillus, Proteus, Salmonella and Streptococcus faecalis and others, has been observed in mixtures obtained by heating arginine and xylose or histidine and glucose (Einarsson et al, 1983, 1988). An interesting topic, still to be investigated in detail, is the relationship of MRPs with products that derive from physiological protein glycation, especially in diabetic patients and that are involved in ageing and act as promoting agents in Alzheimer’s disease. However, it has been suggested that dietary restriction of food MRPs may be useful to reduce the burden of AGEs in diabetic patients and possibly improve the prognosis of the disease (Koschinsky et al, 1997). 11.7 Formation of toxic compounds Epidemiological studies indicate that diet is an important factor in human cancer. In this respect, the negative consequence of thermal processing is the possible formation of highly mutagenic heterocyclic amines (HAs), that have attracted a growing interest since their discovery about 25 years ago (Sugimura et al, 1977). HAs include about 20 different derivatives that are found at ppb level in cooked muscle foods and can be divided in two classes: the amino-carbolines and the amino-imidazo-azaarenes (AIAs). Amino-carbolines are sometimes called pyrolysis products, because they were first isolated from smoke condensates collected from cigarettes or from pyrolysed single amino acids or proteins. Figure 11.14 shows the structures of 282 The nutrition handbook for food processors the carbolines that have been detected in food or model systems. A typical feature is the presence of an exocyclic amino group on the pyridine ring, with the excep- tion of b-carbolines (harman and norharman) that do not have the amino group and are not mutagenic, but have been demonstrated to be comutagenic (Hatch, 1986). They are assumed to be formed by a free-radical mechanism, but the path- ways of their formation are still rather obscure. The a- and b-carbolines are formed by pyrolysis of animal proteins, such as albumin and casein, as well as of vegetable proteins, for example soy protein isolates. They are formed in model systems at temperatures similar to those applied to food cooking (J?gerstad et al, 1998). b-Carboline concentration in foods is generally 10–100 times greater than in that of other more mutagenic congeners. AIAs are also referred to as thermic mutagens because they are formed at temperatures used during ordinary cooking and were first isolated from cooked meat and fish. Since they are very mutagenic, much attention has been applied to their determination in foods. They are characterised by the presence of a 2- amino-imidazo group, indispensable for genotoxic/mutagenic activity, and derive from the condensation of creatine with an aldehyde, coming from the Strecker degradation, and a pyrazine or pyridine. There are indications that free radicals may be involved (Pearson et al, 1992). Phenylalanine and creatine are the pre- cursors of PhIP (Felton and Knize, 1991). The number and position of the methyl groups on each ring contributes to a wide number of congeners (Fig. 11.15): the 2-amino-imidazo group is indis- pensable for mutagenic activity, but the number and position of the methyl groups determine the genotoxic potential of each congener (Benigni et al, 2000). They are fairly stable under ordinary cooking conditions, but start to degrade under prolonged heating (Arvidsson et al, 1997). A very detailed recent review (J?gerstad et al, 1998) contains a complete list of these toxic compounds and of the foods where they may be encountered. Minimising their formation requires knowledge of precursors, cooking conditions, reaction mechanisms and kinetics as well as information that the food industry needs to choose optimal Thermal processing and nutritional quality 283 N H N NH 2 N H N N H N NH 2 N H N alpha-carbolines gamma-carbolines beta-carbolines delta-carbolines N H N NH 2 CH 3 N H N CH 3 H 3 C N H N NH 2 H 3 C CH 3 NH 2 N H N NH 2 H 3 C AC MeAC Harman Norharman Trp-P-2 Trp-P-1 Glu-P-1 Glu-P-2 Fig. 11.14 Structures and trivial names of amino-carbolines. conditions for designing food processes and food processing equipment. Tem- perature and cooking time are relevant parameters in determining their amount, and of the two temperature is the more critical (Knize et al, 1994). A kinetic model for the formation of polar HAs in a meat model system has been proposed (Arvidsson et al, 1998) and a detailed discussion of the selection of conditions and additives that may minimise their formation may be found in J?gerstad et al (1998). HAs are found mostly in the crust of grilled and fried meat and fish and in the pan residue and, to a much lesser extent, in the interior of meat (Skog et al, 1995). In some countries pan residues are used to make gravy, which may result in a substantial contribution of HAs to the diet; to discard the pan residue is certainly a highly recommended habit. The low concentration of HAs and the complex sample matrix of cooked foods make the analysis of these compounds very difficult (Pais and Knize, 2000). Most data reported in the literature refer to polar HAs in cooked muscle foods that are found at low ng/g level (for reviews see Skog, 1993; Layton et al, 1995). An improved method for the determination of non-polar HAs has been published more recently (Skog et al, 1998). Many HAs have been shown to be carcinogenic in mice, rats, and non-human primates. However, epidemiological studies show conflicting data: some have shown an association between cooked meat and fish intake and cancer develop- ment and others no significant relationship (Sugimura et al, 1993; Steineck et al, 1993). Human liver metabolically activates some HAs through cytochrome P450 mediated N-oxidation and subsequent esterification reactions to produce the ulti- 284 The nutrition handbook for food processors N N N CH 3 NH 2 R 1 N N N N CH 3 NH 2 R 1 R 2 R 3 R 1 = H IQ R 1 =CH 3 MeIQ R 1 = R 2 = R 3 = H IQx R 1 = CH 3 , R 2 = R 3 = H MeIQx R 1 = CH 3 , R 2 = H, R 3 = CH 3 4,8-DiMeIQx R 1 = CH 3 , R 2 = CH 3 , R 3 = H 7,8-DiMeIQx R 1 = R 2 = R 3 = CH 3 4,7,8-TriMeIQx R 1 = CH 3 , R 2 = H, R 3 = CH 2 OH 4-CH 2 OH-8-MeIQx N N N N H 3 C CH 3 CH 3 NH 2 N N N CH 3 NH 2 H 3 C N N N CH 3 NH 2 R R 7,9-DiMeIgQx R = H 1,6-DMIP R = CH 3 1,5,6-TMIP R = H PhIP R = OH 4'-OH-PhIP Fig. 11.15 Structures and trivial names of aminoimidazo-azaarenes. mate carcinogenic metabolites (Friedman, 1996). This metabolic activation leads to DNA adducts (Schut and Snyderwine, 1999). The International Agency for Research on Cancer (IARC) regards some HAs as possibly or probably carcinogenic to humans and recommends to minimise exposure to them (IARC, 1993). Data suggest that HAs are the only known animal colon carcinogens that humans other than vegetarians consume every day and, although very difficult, it would be desirable to control their level in food. A detailed overview of the risk assessment can be found in a review by Friedman (1996). 11.8 Future trends From the point of view of nutrition the two most important areas of research are either the toxicological aspects, principally related to the formation in particular conditions of mutagenic heterocyclic amines, or the positive nutritional features of some MRPs. In this sense most of the efforts are toward a better comprehen- sion of the role of melanoidins in health, especially in relation to their antioxi- dant properties. In fact, in recent years, nutritionists have dedicated much interest to the pres- ence of antioxidants in vegetable foods. Some traditional products, such as extra virgin olive oil, red wine and tomato, have received great promotion from a better comprehension of their beneficial role in diet. Tomato is an interesting case because it has been demonstrated that in contrast to what one might superficially expect processing enhances the antioxidant activity (Anese et al, 1999). This fact has opened completely new scenarios to the possibilities offered in this field by processed foods. The European Commission is financing a European cooperation in the field of scientific and technical research: COST Action 919 ‘Melanoidins in Food and Health’, which aims to promote research on melanoidins both in food and health. The two work packages of greatest interest are WP4 ‘Antioxidant and other prop- erties related to food shelf-life’, and WP5 ‘Effects on health as assessed by in vitro and in vitro studies’. The results are being published in a series of books (Ames, 2001). 11.9 Sources of further information and advice The reader will find useful discussion about the thermal treatment of food in Belitz and Grosch (1999). Every three to four years researchers working on the MR both in food science and medicine meet for an extremely important interna- tional symposium, whose proceedings are a very useful source of multidiscipli- nary information (Waller and Feather, 1983; Fujimaki et al, 1986; Finot et al, 1990; Labuza et al, 1994; O’Brien et al, 1998). 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