Addeo F, Garro G, Intorcia N, Pellegrino L, Resmini P, Chianese L
Dipartimento di Scienza degli Alimenti, Facoltà di Agraria, Università degli Studi di Napoli Federico II, Portici, Italia.
J Dairy Res. 1995 May;62(2):297-309. doi: 10.1017/s0022029900030995.
The whole N fraction of six samples of hard and semi-hard pressed cheeses was analysed using PAGE, polyacrylamide gel isoelectric focusing and immunoblotting with polyclonal antibodies against beta- and alpha s1-casein. The origin of some electrophoretic bands corresponding to peptides produced from the enzymic degradation of the casein fractions was established. A number of these peptides were also present in the in vitro hydrolysates of casein with plasmin and chymosin. Thus, it was also possible to determine which casein was the source of each peptide and which enzymes were active in cheese. Compared with the traditional Coomassie staining procedures, immunoblotting is more sensitive and specific, making the interpretation of each electrophoretic profile easy. Thus, it was also possible to obtain a clear picture of the state of each casein fraction in a cheese variety. Two main peptides were isolated from the pH 4.6-insoluble N fraction of Parmigiano-Reggiano using DEAE-cellulose chromatography and identified, from the amino acid sequence of the N- and C-terminal ends, as gamma 3-casein (beta-casein(f108-209)) and alpha s1-PL1 (alpha s1-casein(f80-199). In both cases, a Lys-X bond was hydrolysed, indicating the action of a trypsin-like enzyme in beta- and alpha s1-casein hydrolysis during the ripening of this variety of hard pressed cheese.
使用聚丙烯酰胺凝胶电泳（PAGE）、聚丙烯酰胺凝胶等电聚焦和针对β-酪蛋白和αs1-酪蛋白的多克隆抗体进行免疫印迹分析了六种硬压和半硬压奶酪样品的总氮部分。确定了一些与酪蛋白部分酶解产生的肽相对应的电泳条带的来源。其中许多肽也存在于酪蛋白与纤溶酶和凝乳酶的体外水解产物中。因此，还可以确定每种肽的酪蛋白来源以及奶酪中哪些酶具有活性。与传统的考马斯亮蓝染色程序相比，免疫印迹更灵敏、更具特异性，使得对每个电泳图谱的解读变得容易。因此，也能够清楚地了解某一奶酪品种中每种酪蛋白部分的状态。使用DEAE-纤维素色谱从帕尔马干酪的pH 4.6不溶性氮部分中分离出两种主要肽，并根据N端和C端的氨基酸序列鉴定为γ3-酪蛋白（β-酪蛋白（f108-209））和αs1-PL1（αs1-酪蛋白（f80-199））。在这两种情况下，一个赖氨酸-X键被水解，表明在这种硬压奶酪成熟过程中，一种类胰蛋白酶在β-酪蛋白和αs1-酪蛋白水解中起作用。
