Bosch Lourdes, Sanz Maria Luz, Montilla Antonia, Alegría Amparo, Farré Rosaura, del Castillo María Dolores
Nutrición y Bromatología, Facultad de Farmacia, Universidad de Valencia, Avda. Vicente Andrés Estellés s/n, 46100 Burjassot, Valencia, Spain.
J Chromatogr B Analyt Technol Biomed Life Sci. 2007 Dec 1;860(1):69-77. doi: 10.1016/j.jchromb.2007.10.011. Epub 2007 Oct 22.
Protein quality was assayed by simultaneous measurement of lysine (Lys), carboxymethyllysine (CML) and lysinoalanine (LAL). GC-FID analysis of N-tert-butyl dimethylsilyl (tBDMSi) derivatives of these amino acids was undertaken. tBDMSi derivates were separated on a CP-SIL 5CB commercially fused silica capillary column (25 m x 0.25 mm i.d., 0.25 microm film thickness) employing a thermal gradient programmed from 200 to 300 degrees C. The identity of tBDMSi derivatives of Lys, CML and LAL was established by GC-MS while FID detection was employed for quantification. Analytical parameters such as linearity (lysine 350-4200 microM, LAL 3-81 microM, CML 16-172 microM), precision (1-13% variation coefficients), accuracy (85-108% average recovery) and limits of detection (lysine 0.4 mg/100 g protein, LAL 5.0 mg/100 g protein, CML 3.4 mg/100 g protein) and quantification (lysine 1.4 mg/100g protein, LAL 15.2 mg/100 g protein, CML 11.2 mg/100 g protein) were determined for validation of the analytical approach. Model systems and real foods have been studied. Kinetic of CML formation from different food proteins (BSA, soy protein, casein and gluten) was performed employing model systems. Carboxymethylation rate depended on the source of protein. Maillard reaction progressed to advanced stages damaging the protein quality of stored infant foods, soy drinks, boiled eggs and dry powdered crepes. CML values ranged from 62 to 440 mg/100 g protein were measured. LAL was also formed during boiling eggs (21-68 mg/100g protein) indicating additional damage by crosslinking reaction. In agreement, lysine content was affected by both food processing and storage.
通过同时测量赖氨酸（Lys）、羧甲基赖氨酸（CML）和赖氨酰丙氨酸（LAL）来测定蛋白质质量。对这些氨基酸的N-叔丁基二甲基硅烷基（tBDMSi）衍生物进行气相色谱-火焰离子化检测（GC-FID）分析。tBDMSi衍生物在CP-SIL 5CB商业熔融石英毛细管柱（25 m×0.25 mm内径，0.25微米膜厚）上分离，采用从200℃到300℃的程序升温。Lys、CML和LAL的tBDMSi衍生物的身份通过气相色谱-质谱联用（GC-MS）确定，同时采用火焰离子化检测（FID）进行定量。确定了线性（赖氨酸350 - 4200 microM、LAL 3 - 81 microM、CML 16 - 172 microM）、精密度（变异系数1 - 13%）、准确度（平均回收率85 - 108%）以及检测限（赖氨酸0.4 mg/100 g蛋白质、LAL 5.0 mg/100 g蛋白质、CML 3.4 mg/100 g蛋白质）和定量限（赖氨酸1.4 mg/100g蛋白质、LAL 15.2 mg/100 g蛋白质、CML 11.2 mg/100 g蛋白质）等分析参数，以验证该分析方法。对模型系统和实际食品进行了研究。利用模型系统研究了不同食品蛋白质（牛血清白蛋白、大豆蛋白、酪蛋白和面筋）形成CML的动力学。羧甲基化速率取决于蛋白质来源。美拉德反应进展到高级阶段，损害了储存的婴儿食品、大豆饮料、煮鸡蛋和干薄饼的蛋白质质量。测得的CML值范围为62至440 mg/100 g蛋白质。煮鸡蛋过程中也会形成LAL（21 - 68 mg/100g蛋白质），表明交联反应造成了额外损害。与此一致的是，赖氨酸含量受到食品加工和储存的影响。
