Transformations not involving sugars crosslinked 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 normally indicated with the trivial name of lysinoalanine (LAL) (Maga, 1984). Analogous reactions may involve ornithine to give ornithinoalanine (OAL), cysteine to give lanthionine (LAN), and histidine to produce histidinoalanine (HAL)

oH^H dehydroalanine protein elimination

Z = OPO3H2, phosphoserine = O-glycoside, glycoserine = S-S-CH2CH(NH2)COOH, cystine lysine lysinoalanine

Fig. 11.12 Mechanism of the formation of dehydroalanine residues in protein chains.


,CH2-(CH2)3-CH-COOH ,CH2"(CH2)2-CH-COOH HN * * HN * *

nch2-ch-cooh nch2-ch-cooh Nh2 lal nh2 oal h2c




Fig. 11.13 Main cross-linker amino acids.

(Finley and Friedman, 1977). Both nitrogens of histidine may react, giving rise to the regioisomers N^-HAL and NT-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 compounds that derive from modification of the amino acid side chains in proteins, such as isopeptides, must be considered. This makes them useful in quality control as molecular markers of the processes applied in food preparation (Pellegrino 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 products 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 microprotein 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 150-2120 mg/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 80 mg/g protein in adapted formulas, 80-370 mg/g protein in follow-on formulas, and about 500 mg/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 800 mg/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 (pKa = 5.5) in respect to e-NH3+ of lysine (pKa = 10).

Analysis of various milk-protein containing foods, such as heated skim milk, sterilised milk, and baby formulas, permitted detection of amounts of HAL between 50 and 1800 mg/g protein, comparable to LAL amounts (Henle et al, 1993; Henle et al, 1996). However, no toxicological effects have been reported for HAL.

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