D-alanine (D-Ala), D-aspartic acid (D-Asp) and D-glutamic acid (D-Glu) are important constituents of the cell walls of bacteria responsible for fermentation processes, and have been detected in several fermented foods, such as cheeses, yogurt and vinegar (Palla et al. 1989; Brückner & Hausch, 1990; Dossena et al. 1991; Brückner et al. 1992; Gandolfi et al. 1992). In particular, D-amino acids have been found, free and abundant, in cheeses requiring long ripening periods (1–2 years; Santaguida et al. 1995). Their presence in cheeses could be related mainly to lysis of the bacterial wall, to which short-chain D-amino acids are bound, but also to the activity of bacterial racemases (Adams, 1972), which convert free L-amino acids generated during proteolysis. The content of these D-amino acids and the D:L ratio have been recently proposed as indicators of ripening and for quality assessment of Parmigiano Reggiano and Grana Padano cheeses (Marchelli et al. 1997).

The aim of the present study was to determine D-amino acids in cheeses with shorter ripening periods to evaluate whether they could be useful as indicators of ripening and for quality assessment. The work was carried out with Montasio, a typical cheese produced in north-east Italy using traditional methods. Montasio is a semi-hard cheese produced from unpasteurized milk, cooked at 44–46 °C and consumed after at least 2 months ripening. This study represents an extension of previous work on free amino acid contents and ripening in semi-hard cheeses (Innocente, 1997).

As the demands for food products of high quality increase, it will become more important to be able to modify the properties of food proteins. One possibility is to use enzymic modifications to improve functional properties and nutritional values. A highly efficient class of enzymic crosslinkers are transglutaminases (TG, EC 2.3.2.13), calcium-dependent enzymes that catalyse an acyl transfer reaction between protein-bound glutaminyl residues and primary amines (for review, see Aeschlimann & Paulsson, 1994). When protein-bound lysyl residues act as acyl acceptors, the reaction leads to the formation of intramolecular and/or intermolecular isopeptide bonds. Although a wide variety of amines such as putrescine, cadaverine or lysyl residues may act as amine donors in this reaction, only a limited number of glutamine residues in certain proteins will act as amine acceptors (Gorman & Folk, 1980).

Ikura et al. (1981, 1985) reported that TG can be used to introduce methionine into casein and soyabean proteins and lysine into wheat gluten, thereby showing that TG can also be utilized to improve the nutritional value of food proteins. There have been a number of reports concerning the TG-mediated polymerization of food proteins such as α-lactalbumin and β-lactoglobulin (Aboumahmoud & Savello, 1990; Færgemand et al. 1997), soyabean proteins and casein components (Ikura et al. 1980a, b) and pea legumin (Larré et al. 1993). Recently, improved protein foaming capacity and stability have been demonstrated in TG-catalysed polymers of soyabean protein and whey protein isolate (Yildirim et al. 1996).

Bovine PP3 (for review, see Girardet & Linden, 1996) is a phosphorylated glycoprotein isolated from the proteose peptone fraction of milk (Sørensen & Petersen, 1993a). The primary structure of PP3 has been determined (Sørensen & Petersen, 1993b) and comprises a polypeptide backbone of 135 amino acid residues containing five phosphorylated serines, two threonine-linked O-glycosylations, and one N-glycosylation. Immunological studies have shown that PP3 is present in the milk fat globule membrane, and that PP3 forms multimeric aggregates in bovine milk (Sørensen et al. 1997). Several functions have been suggested and investigated for bovine PP3 and fractions enriched with PP3, including emulsification (Shimizu et al. 1989), inhibition of lipolysis (Girardet et al. 1993) and mitogenesis (Mati et al. 1993).

Previously, we have localized the potential TG-reactive glutamines in the four bovine caseins (Christensen et al. 1996) and in milk osteopontin (Sørensen et al. 1994). In the present study we have shown that component PP3 is a substrate for guinea-pig liver TG containing both reactive glutamine and lysine residues. In addition, we have localized the glutamine residues that act as amine acceptors.

Brucellosis can be transmitted to man by direct contact with infected animals or through contaminated meat, milk and dairy products (Nicoletti, 1989). The analysis of Brucella spp. is carried out in the laboratory by microbiological or serological assays (Alton et al. 1988). The first are more specific but are also time-consuming and expose the analyst to the risk of infection (López-Merino, 1991). However, the latter can result in false positives owing to cross reactivity with other Gram-negative bacteria (Diaz-Aparicio et al. 1994). Because of these limitations, the amplification in vitro of specific DNA regions by the polymerase chain reaction (PCR) could represent a powerful tool for rapid and specific diagnostic analysis. In recent years, several PCR methods have been developed to amplify specific DNA sequences of Brucella strains (Herman & de Ridder, 1992; Romero et al. 1995; Valentino et al. 1997). In addition, direct analysis of Brucella in contaminated abortive tissues (Fekete et al. 1992), milk and blood (Leal-Klevezas et al. 1995; Rijpens et al. 1996) has been reported.

In this paper we describe a method for gene-specific PCR amplification of a 443 base pair (bp) fragment of Brucella DNA that belongs to a gene encoding for a 31 kDa outer membrane protein. This protein (BCSP-31) is a membrane antigen characteristic of the Brucella genus (Mayfield et al. 1988). The PCR method was developed for the analysis of soft cheeses. We focused our attention on Mozzarella, Pecorino and ricotta samples, because such products are not subjected to the natural microbial autopurification process of maturing. They are widely consumed in Italy and a relationship between infected foods and the areas where brucellosis is a human zoonosis is a possibility.

The analysis was performed without purification of DNA from bacteria. Indeed, after homogenization, the sample was subjected to thermal shock by freeze–thaw cycles that lysed bacteria and solubilized nucleic acids for subsequent PCR amplification. Amplified DNA fragments were separated by agarose gel electrophoresis and visualized by ethidium bromide staining. Several brands of soft cheeses and ricotta contaminated at different levels with Brucella cells were analysed by our procedure to evaluate the detection sensitivity and the repeatability of the method.

The composition and sensory characteristics of matured cheeses are controlled by a number of factors, among which the type of feed is important. The influence of feeding can be reflected by the presence in cheeses of terpenes and sesquiterpenes, compounds typically indicating their vegetable origin (Mariaca et al. 1997). Indeed, several investigators have already established that these compounds could characterize the forage even to a specific geographical location. Dumont & Adda (1978), Dumont et al. (1981), Guichard et al. (1987), Bosset et al. (1994) and Moio et al. (1996) were thus able to distinguish cheeses from lowland and upland regions. In most of these studies, the conditions under which milk was produced and processed were not completely controlled. Furthermore, all these studies analysed only the volatile fraction of cheeses and did not examine the volatile compounds in the forages used. Recently Bosset et al. (1994), in one part of the project described by Jeangros et al. (1997), showed that highland grass with a highly diversified botanical composition produces milk and cheeses with significantly different chemical compositions from those from lowland grass. To improve our understanding of the relationship between animal feed and cheese composition, we have investigated under controlled experimental conditions both the composition of the terpene and sesquiterpene fractions of four forages with different botanical diversities and the influence of those forages on the terpene fraction of cheeses.

Kefir is a refreshing fermented milk with a slightly acidic taste obtained by incubating milk with kefir grains (Saloff-Coste, 1996). Kefir grains are a complex mixture of lactic acid bacteria and yeasts in a strong and specific association. They are characterized by an irregular form, a folded and uneven surface and a white or slightly yellow colour. They are tough and resilient and have a characteristic acid taste (Bottazzi et al. 1994). The basic microflora contains lactococci, homofermentative and heterofermentative lactobacilli, yeasts and acetic acid bacteria (Bottazzi et al. 1994; Rea et al. 1996). Among the yeasts isolated from grains and identified are Candida kefir, Saccharomyces cerevisiae, Sac. delbrueckii, Cand. holmii, Sac. unisporus and Sac. lipolytica (Angulo et al. 1993; Marshall, 1993; Garrote et al. 1997). Lactobacillus brevis, Lb. viridescens, Lb. casei, Lb. kefir, Lb. kefiranofaciens, Lb. kefirgranum, Lb. parakefir, Leuconostoc spp. and Lactococcus lactis are among the lactic acid bacteria present in the grains (Marshall et al. 1984; Toba et al. 1991; Takizawa et al. 1994; Garrote et al. 1997). The mixed microflora of yeasts and bacteria is held together by a matrix containing (g/kg dry weight) protein 340, polysaccharide 470 (Ottogalli et al. 1973).

The study of kefir grains in milk has been centred on the characteristics of the polysaccharide produced by lactobacilli within the grain (Yokoi et al. 1991). This polysaccharide, named kefiran, is composed of glucose and galactose (Yokoi et al. 1991). It has been suggested that proteins are incorporated from the growth media (Bassette & Acosta 1988), but no details about structure and composition are available.

Soyabeans are an important component of the diet in many countries and have been used to obtain fermented products such as sogurt (Mann, 1991). Special attention has been given to the growth of, and sugar utilization by, Bifidobacterium spp., Lb. delbrueckii subsp. bulgaricus and Streptococcus thermophilus in soya milk (Buono et al. 1990; Murti et al. 1993; Ankenman Granata & Morr, 1996). To our knowledge, there have been no attempts to ferment soya milk with kefir grains.

The aim of this study was to investigate the growth of kefir grains in soya milk and the composition of these grains, focusing on the matrix proteins.

Dulce de leche is a dairy-based confectionery product, widely consumed in Argentina and other Latin American countries as filling or topping for desserts, cakes and biscuits (Hansen, 1978; Bouzas, 1997). It is prepared by heat concentration of whole milk with added sucrose until it reaches 700 g total solids/kg dulce de leche. Sucrose is usually partly replaced by other sugars (not more than 400 g/kg of total sugars added according to Normas Mercosur, 1996) to avoid crystallization. Sodium bicarbonate is added to prevent coagulation of proteins as the pH of the mixture decreases and the isoelectric pH of proteins increases during heating.

Maillard browning is the main reaction of milk proteins during the manufacture of dulce de leche. This results in an attractive flavour and colour, but also leads to less desirable changes, such as loss of nutritional value and formation of toxic compounds (Rizzi, 1994; Friedman, 1996a). The natural reactants are free amino groups of milk proteins and lactose. However, as the addition of other reducing sugars is an accepted practice, the consequences may be quite different depending on the formulation used. In addition, other reactions leading to crosslinkages between protein chains contribute to the reduction in nutritional value (Mauron, 1990; Friedman, 1996b; Finot, 1997). The reduction of nutritional quality of milk proteins is attributed to the lower digestibility and the biological inactivation or destruction of essential amino acids. Lysine is the essential amino acid most affected as its free ε-amino group is very reactive (Hurrell, 1990; Mauron, 1990; Finot, 1997).

Several studies have investigated the physical and rheological characteristics (Pauletti et al. 1984, 1996), sensory properties (Sabioni et al. 1984; Buera et al. 1990; Hough et al. 1990), sugar composition and microbiological stability (Ferramondo et al. 1984) of dulce de leche. However, there is little information about the loss in available lysine during manufacture (Pavlovic et al. 1994).

The aim of this study was to examine the effects of the nature and quantity of various sugars commonly used in the manufacture of dulce de leche on the loss of available lysine.