192 M.V. Moreno-Arribas et al.
and bacteria (Feuillat et al. 1977; Alexandre et al. 2001; Manca de Nadra et al. 2005;
Remize et al. 2006). The main reason for the dearth of studies on this fraction is due
to the complexity and lack of specificity of the techniques used for its analysis.
6B.2 Analytical Methods
6B.2.1 Spectrophotometric Methods
The N-terminalα-amino groups on peptides react quantitatively with reagents such
as ninhydrin (Doi et al. 1981) to form colored derivatives or witho-phthaldialdehyde
(OPA) (Acedo et al. 1994) and fluorescamine to form fluorescent derivatives. These
reactions are useful in making quantitativedeterminations of oligopeptides in wines
and for their detection during chromatographic analysis. Polypeptide content can be
determined by assaying the aromatic amino acids tyrosine and tryptophan, which is
readily achieved by measuring absorbance at 280 nm or by measuring the color
reactions of these amino acids with certain reagents like Folin-Ciocalteu. These
methods are only applicable for the analysis of wine peptides after long stages
of purification, owing to the presence offree amino acids, derivates in the case
of OPA, and the presence of phenolic compounds, which also absorb at 280 nm
and react with Folin-Ciocalteu reagent. These methods do not provide information
regarding the quantity and the nature of peptides. For this purpose, chromatographic
or electrophoretic techniques are required.
6B.2.2 Sample Preparation for Peptide Analysis
Analytical determination of peptides in wine requires sample preparation, involv-
ing their isolation from the remaining components, mainly high molecular weight
nitrogen compounds, free amino acids and phenols. Table 6B.1 summarizes the pro-
cedures used in the literature for the extraction of wine peptides before their analysis
by different analytical techniques and with different detection systems.
The traditional method used to remove proteins from a sample to be analyzed
is to precipitate them using different precipitants obtaining a soluble fraction after
centrifugation. The most common precipitants that have been used are 7% TCA
(trichloroacetic acid) (Yokotsuka et al. 1975) and 95% ethanol (Moreno-Arribas
et al. 1996, 1998a,b; Bartolom ́e et al. 1997; Mart ́ınez-Rodr ́ıguez et al. 2002). Ultra-
filtration has been used as an initial step for isolation of the peptide fraction of
wines by many authors (Usseglio-Tomasset and Di Stefano 1978; Colagrande and
Silva 1981; Acedo et al. 1994; Mart ́ınez-Rodriguez and Polo 2000; Dos Santos
et al. 2000; Desportes et al. 2000, 2001; Person et al. 2004; Pozo-Bay ́on et al. 2005,
Alcaide-Hidalgo et al. 2007). The pore size of the membranes varies in the different
works (from 10,000 to 200–300 Da). To obtain fractions of low relative molecular
mass (less than 500Da) it is recommended to use membranes with an increase in