Jensen Hanne B, Pedersen Katrine S, Johansen Lene B, Poulsen Nina A, Bakman Mette, Chatterton Dereck E W, Larsen Lotte B
Department of Food Science, Aarhus University, 8830 Tjele, Denmark.
Department of Food Science, University of Copenhagen, 1958 Frederiksberg C, Denmark.
J Dairy Sci. 2015 Feb;98(2):747-58. doi: 10.3168/jds.2014-8678. Epub 2014 Dec 12.
Chymosin-induced cleavage of κ-casein (κ-CN) occurs during the first enzymatic phase in milk coagulation during cheese manufacturing, where the hydrophilic C-terminal peptide of κ-CN, named caseino-macropeptide (CMP), is released into the whey. The CMP peptide is known for its rather heterogeneous composition with respect to both genetic variation and multiple posttranslational modifications, including phosphorylation and O-linked glycosylation. An approach of liquid chromatography coupled with mass spectrometry was used to investigate (1) the overall protein profile and (2) the release of various forms of CMP after addition of chymosin to individual cow milk samples from 2 breeds, Danish Jersey (DJ) and Danish Holstein-Friesian (DH). The cows were selected to represent distinct homo- and heterozygous types of the κ-CN genetic variants A, B, and E (i.e., genotypes AA, BB, AB, EE, and AE). Initially, investigation of the protein profile showed milk with κ-CN BB exhibited the highest relative content of κ-CN, whereas AE milk exhibited the lowest, and after 40min of renneting >90% of intact κ-CN was hydrolyzed by chymosin in milk representing all κ-CN genotype. By in-depth analysis of the CMP chromatographic profile, multiple CMP isoforms with 1 to 3 O-linked glycans (1-3 G) and 1 to 3 phosphate groups (1-3 P) were identified, as well as nonmodified CMP isoforms. The number of identified CMP isoforms varied to some extent between breeds (21CMP isoforms identified in DJ, 26CMP isoforms in DH) and between κ-CN genetic variants (CMP variant A being the most heterogeneous compared with CMP B and E), as well as between individual samples within each breed. The predominant forms of glycans attached to CMP were found to be the acidic tetrasaccharide {N-acetyl-neuraminic acid α(2-3)galactose β(1-3)[N-acetyl-neuraminic acid α(2-6)]N-acetyl galactose} or trisaccharides {N-acetyl-neuraminic acid α(2-3)galactose β(1-3)N-acetyl galactose and galactose β(1-3)[N-acetyl-neuraminic acid (α2-6)]N-acetyl galactose}. The CMP release was calculated to follow first-order kinetics and was determined by the measurement of CMP content during renneting. The highest rate of release for all CMP isoforms occurred from 0 to 2min after chymosin addition. Concurring results from both breeds showed that CMP variant A with 1-2 P had the highest reaction rate of CMP release, followed by CMP B 1-2 P and then by CMP E 1-2 P (only in DH). All the identified glycosylated CMP isoforms had lower reaction rates of release compared with that of nonglycosylated CMP, thus glycan modifications seemed to negatively influence the reaction rate of chymosin-induced hydrolysis of κ-CN.
在奶酪制造过程中，凝乳酶诱导的κ-酪蛋白（κ-CN）裂解发生在牛奶凝固的第一个酶促阶段，在此过程中，κ-CN的亲水性C末端肽，即酪蛋白巨肽（CMP），被释放到乳清中。CMP肽因其在遗传变异和多种翻译后修饰（包括磷酸化和O-连接糖基化）方面相当不均一的组成而闻名。采用液相色谱与质谱联用的方法，研究了（1）整体蛋白质谱，以及（2）向来自丹麦泽西牛（DJ）和丹麦荷斯坦-弗里生牛（DH）这两个品种的个体牛奶样品中添加凝乳酶后各种形式CMP的释放情况。选择这些奶牛代表κ-CN基因变体A、B和E的不同纯合和杂合类型（即基因型AA、BB、AB、EE和AE）。最初，对蛋白质谱的研究表明，含κ-CN BB的牛奶中κ-CN的相对含量最高，而含AE的牛奶中κ-CN的相对含量最低，并且在凝乳40分钟后，代表所有κ-CN基因型的牛奶中>90%的完整κ-CN被凝乳酶水解。通过对CMP色谱图的深入分析，鉴定出了具有1至3个O-连接聚糖（1-3 G）和1至3个磷酸基团（1-3 P）的多种CMP异构体，以及未修饰的CMP异构体。鉴定出的CMP异构体数量在品种之间（DJ中鉴定出21种CMP异构体，DH中鉴定出26种CMP异构体）、κ-CN基因变体之间（与CMP B和E相比，CMP变体A最为不均一）以及每个品种内的个体样品之间存在一定程度的差异。发现附着在CMP上的聚糖的主要形式是酸性四糖{N-乙酰神经氨酸α(2-3)半乳糖β(1-3)[N-乙酰神经氨酸α(2-6)]N-乙酰半乳糖}或三糖{N-乙酰神经氨酸α(2-3)半乳糖β(1-3)N-乙酰半乳糖和半乳糖β(1-3)[N-乙酰神经氨酸(α2-6)]N-乙酰半乳糖}。计算得出CMP的释放遵循一级动力学，并通过在凝乳过程中测量CMP含量来确定。所有CMP异构体的最高释放速率发生在添加凝乳酶后的0至2分钟。两个品种的一致结果表明，具有1-2个P的CMP变体A的CMP释放反应速率最高，其次是具有1-2个P的CMP B，然后是具有1-2个P的CMP E（仅在DH中）。与未糖基化的CMP相比，所有鉴定出的糖基化CMP异构体的释放反应速率较低，因此聚糖修饰似乎对凝乳酶诱导的κ-CN水解反应速率有负面影响。
