**4. Analyses of Maillard reaction products**

MRPs are present in the diet and many authors have highlighted their health benefits and risks. For that reason, it is of most importance characterizing and quantifying MRPs in common foods, to get the best balance between benefits and potential risks, and then to establish guidelines for food health (Delgado-Andrade *et al.,* 2009).

Development of sophisticated analytical techniques made it possible to isolate, characterize, and quantify several specific non browning reaction compounds formed *in vitro* and *in vivo,*  both at the early and advanced stages of Maillard reaction. Among them the most common are: Amadori compounds (indirectly analyzed as furosine), Nε-(carboxymethyl)lysine (CML) and some intermediate derivatives of the reaction, such as hydroxymethylfurfural. Measurement of fluorescent compounds formed during the reaction is also a reliable tool to evaluate the extension and ratio of nutritional loss due to thermal processing of foods (Delgado-Andrade *et al.*, 2009; Friedman, 1996).

#### **4.1 Fluorescent compounds**

286 Food Industrial Processes – Methods and Equipment

The volatile products of the Maillard reaction can be classified according to the sugar dehydration/fragmentation products as furans, pyrones, cyclopentenes, carbonyls and acids; the amino acid degradation products as aldehydes and sulfur compounds; and the volatiles produced by further interactions as pyrroles, pyridines, imidazoles, pyrazines, oxazoles, thiazoles, and others. Pyrazines and alkylpyrazines are associated with the flavor and aroma of cooked (roasted) and nutty, respectively. Alkylpyridines confer to foods flavor and aroma of green, bitter, astringent and burnt, and furans, furanones and pyranones of

Compounds that are essential to the characteristic flavor and aroma of food products are generally present at trace levels. The oxygen-containing aroma compounds 2,3-butanedione, 2,3-pentanedione, methylpropanal, 3-methylbutanal, phenylacetaldehyde, 3-hydroxy-4,5 dimethyl-2(3*H*) furanone and 2,5-dimethyl- 4-hydroxy-3(2*H*)-furanone occur in concentration ranging from 1μg/kg up to 100 mg/kg. The nitrogen-containing aroma compounds 2-ethyl-3,5dimethylpyrazine, 2,3-diethyl-5-methylpyrazine and 2-acetyl-1 pyrroline are present in food in an order of magnitude of 0.001–10 mg/kg. On the whole, sulfur containing Maillard odorants constitute the most powerful aroma compounds and often play, although at trace levels, a dominant role in the flavor of cooked meats. These volatile compounds are responsible for the flavor and aroma to stewed beef juice, boiled trout, french fries, bread crust, cooked chicken, roasted chicken, boiled beef, cocoa powder,

The meat-related flavor compounds are mainly sulphur containing compounds, derived from cysteine and ribose (coming from nucleotides), while the amino acid proline gives rise to typical bread, rice and popcorn flavors. Cysteine-containing mixtures seem to have the most intense meat-like and sulphur smell. The other amino acid-containing sulphur, methionine, generates a highly intense smell of potatoes and it is employed, in the food industry, to enhance the soft flavor of potatoes. Mixtures containing amino acids other than cysteine or methionine in a combination with reducing sugars are characterized mostly by

The food industry invests great effort trying to create synthetic flavors and aromas by reconstituting combinations of these compounds. The process of creating synthetic flavors is limited since the subtleties of flavor perception are many and varied, and although Electronic Noses may detect these compounds, human sensory perception is considered

Texture definition is complex and a general agreement has been reached which evolved from the efforts of a number of researchers. According to Szczesniak (2002), ''texture is the sensory and functional manifestation of the structural, mechanical and surface properties of

Maillard reaction influences the texture of food via protein cross-linking. Manipulation of the extent and nature of such protein cross-linking during food processing offers a means by which the food industry can modify the functional properties of food. Despite of this, the extent of how protein cross-linking affects food texture in processed foods and how to control this parameter to maximize food quality is not yet known (Gerrard, 2002b). Protein cross-linking by the Maillard reaction will affect not only texture, but the protein

sweet, burnt, pungent and caramel-like flavors/aromas.

peanuts, pilsner, roasted beef, popcorn and coffee (Cerny, 2008).

caramel and jammy smell (Stanimirova *et al*., 2011; Van Boekel, 2006).

**3.3 Texture** 

digestibility as well.

essential to validate instrumental data, (Gerrard, 2002a; Schaller et al., 1998).

foods detected through the senses of vision, hearing, touch and kinesthetics''.

Traditionally, Maillard reaction monitoring in food processing was based on the spectrophotometric evaluation of color development at 420 nm. More recently, the evaluation of fluorescent compounds generated by the Amadori rearrangement product undergoing dehydration and fission has become usual. Besides its use in food systems, fluorescence measurement is also employed to evaluate Maillard reaction at physiological conditions, meaning, AGEs generation, and also to access AGEs correlated pathologies development (Delgado-Andrade *et al.,* 2006).

Fluorescent compounds (FC) are precursors for the brown pigments formed in the Maillard reaction, which present different chemical structures. Evaluation changes in fluorescence intensity helps evaluating the extent of the Maillard reaction in food products (Morales & Van Boekel, 1998; Rufiàn-Henares & Delgado-Andrade, 2009) and biological systems. Fluorescence was first used to evaluate the formation of MRPs in milk and now it is used to monitor the processing of cereals, cookies, soybeans, infant formula, cooked salmon and bakery products.

Fluorescent compounds may be free in the matrix or linked to te protein fraction. Total FC (free + linked to protein) determination demands a previous enzymatic hydrolysis, which requires the use of a nonspecific protease (pronase) (Delgado-Andrade *et al*., 2008). Free and total FC have been tested in foods such as milk, breakfast cereals, cooked salmon, roasted soy and enteral formula (Delgado-Andrade *et al.,* 2008; Rufiàn-Henares *et al.,* 2002).

Fluorescence of Advanced Maillard products and Soluble Tryptophan (FAST) is a well established method used to evaluate the nutritional and lysine damages. FAST is based on the quantification of protein denaturation using fluorescence: (1) fluorescent of the advanced Maillard products (FAMP), such as pyrrole and imidazole derivatives, at excitation/emission 330/420 nm; and (2) tryptophan fluorescence (FTrp) at excitation/emission 290/340 nm at pH 4.6. The FAST index is calculated as follows: (100\*FAMP/FTrp) (Birlouez-Aragon *et al.,* 2002).

Furosine is the most specific and important indicator of the initial phase of the Maillard reaction. It is widely used in the analysis of cereal products, since lysine is the limiting amino acid of this product and, thus the presence of furosine is an important marker of protein biological value loss. Monitoring furosine formation and contents helps tailor the processing conditions in order to guarantee the maintenance of the nutritional value of food

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Regarding analytical procedures, in 1992, an ion pairing HPLC based methodology was proposed and successfully applied in a series of studies. In 1996, when furosine became commercially available, Reverse Phase-HPLC became the method of choice for furosine analysis. Due to the possible transformation of CML into furosine during heating it is necessary for the acid hydrolysis to be performed in an inert atmosphere, which impairs

Nε-(carboxymethyl)lysine (CML) is a stable, low reactivity advanced Maillard product (Figure 6). CML can be produced by Amadori compound degradation, such as Nε- (fructosyl)lysine (FL). FL undergoes oxidation to form, Nε-(carboxymethyl)lysine (CML). Rdicarbonyls such as glyoxal (GO), formed during the oxidation of the sugar or the Amadori rearrangement products are immediate precursors of CML. Lipid peroxidation is another route to CML, and GO has been suggested as an intermediate. CML is one of the more important markers of bioactive Maillard products and its content is usually correlated to the

(CH2)4

NH

CH2

The three main metodologies proposed to evaluate CML in foods are: a) RP-HPLC (Reverse Phase/ High Performanc Liquide Chromatography), b) GC/MS (Gas Chromatography connected to Mass Spectrometry) following methylation of the carboxylic group and acylation of the amine group and c) enzyme- linked immunosorbent assay based on a

Currently, CML analyses in foods are performed by specific immunosorbent (AGE-ELISA-Enzyme Linked Immuno Sorbent Assay). This test is suitable for quantitative CML analysis

Among the Maillard reaction products, CML is the best characterized end product. It is employed as an advanced glycation end products/advanced lipoxidation end products

HOOC

R

products (Rufiàn-Henares *et al.*, 2004, 2006; Resmini *et al*., 1990).

health risk of ingestion of heat-treated foods (Charissou *et al.*, 2007).

furosine degradation (Erbersdobler & Somoza, 2007).

**4.4 Nε-(carboxymethyl)lysine** 

Fig. 6. Chemical structure of CML

monoclonal anti-CML antibody (Charissou *et al.*, 2007).

both in biological samples and food (Goldberg, 2004).

(AGEs/ALEs) markers in research (Goldberg, 2004).
