No abstract

Lactuloselysine (e-iV^-deoxylactulosyl-L-lysine) and fructoselysine (e-Ndeoxyfructosyl-L-lysine), formed by Maillard reaction, were identified in enzymic hydrolysates of casein from stored ultra-heat-treated (UHT) milk. Furosine (e-JV-(2-furoylmethyl-L-lysine) and pyridosine (e-(3-hydroxy-4-oxo-6-methyl-l-pyridinyl)-L-norleucine) were identified in the corresponding acid hydrolysates. Enzymic hydrolysis of the caseins, gel filtration and preparative scale amino acid analysis were used to isolate the compounds which were then identified by reference to synthesized authentic compounds. Lactuloselysine formation was extensive and involved 10-30 % of lysine residues in UHT milks stored at 30-37 °C for 6 months to 3 years. Fructoselysine concentration was generally about 10 % of the lactuloselysine concentration.

Casein samples from untreated milk and stored ultra-heat-treated (UHT) milk were hydrolysed with pronase (protease ex Streptomyces griseus K-l) and subsequently with a mixture of peptidases prepared from the microsomal fraction of hog kidneys. Incubation of casein from unheated milk with pronase alone hydrolysed 70-80% of peptide bonds involving He, Leu, Tyr, Phe and His residues; other amino acids were released less well and proline hardly at all. The pronase/peptidase treatment resulted in 90-100% hydrolysis of peptide bonds involving all amino acids, including proline.Caseins from stored UHT milks were more resistant to proteolysis than casein from unheated milk. Reduced release of all amino acids was observed from samples taken after storage at 37 °C for 12 months or longer and for Lys, Arg and Asn residues from samples taken after storage at 30 °C for 14 months. Resistance to proteolysis was attributed to the Maillard reaction between milk proteins and lactose during storage of UHT milk.

Four chemical methods were used in a comparison of the measurement of the extent of modification of lysine residues in casein during storage of ultra-heattreated (UHT) milk. Determination of homoarginine after reaction with o-methylisourea appeared to be the best measure of total lysine modification. Reduction with borohydride before acid hydrolysis prevented recovery of lysine from lactuloselysine (e-iV-deoxylactulosyl-L-lysine) and fructoselysine (e-iV-deoxyfructosyl-L-lysine). This procedure gave values for these derivatives which were in good agreement with those obtained from the determination of furosine (e-iV-(2-furoylmethyl)-L-lysine) in acid hydrolysates. Determination of lysine after acid hydrolysis showed that lysine loss was accounted for by lactuloselysine and fructoselysine formation in most of the samples.Browning was most apparent in UHT milk stored at 37 °C for 3 years and in this sample only part of the lysine residues was accounted for as lactuloselysine and fructoselysine, thus indicating that lysine had become involved further in sugar degradation products.The identification and measurement of lactuloselysine and fructoselysine in the enzymic hydrolysates of caseins from stored ultra-heat-treated (UHT) milk showed that Maillard type interactions of lactose and the e-NH 2 group of lysine take place during storage of UHT milk (Moller, Andrews & Cheeseman, 1977a, b). It was not known, however, to what extent lactuloselysine and fructoselysine accounted for the total modification of lysine in the samples.Hurrell & Carpenter (1974) have studied a number of methods for examining the lysine-content of heat-damaged proteins and related their findings to the nutritional availability of the lysine. They also discussed the considerable differences between results given by the various procedures and concluded that the 2 methods most suitable when studying the involvement of lysine in compounds formed during the early stages of the Maillard reaction were the direct determination of fluorodinitrobenzene-reactive lysine and reaction with o-methylisourea (MIU) (Hurrell & Carpenter, 1974).