通过β-乳球蛋白的反相高效液相色谱法检测和定量受保护原产地名称奶酪中牛、羊和山羊奶的百分比。

Detection and quantification of bovine, ovine and caprine milk percentages in protected denomination of origin cheeses by reversed-phase high-performance liquid chromatography of beta-lactoglobulins.

作者信息

Ferreira Isabel M P L V O, Caçote Helena

机构信息

REQUIMTE, Serviço de Bromatologia, Faculdade de Farmácia, Universidade do Porto, R Aníbal Cunha 164, Porto 4050-047, Portugal.

出版信息

J Chromatogr A. 2003 Oct 10;1015(1-2):111-8. doi: 10.1016/s0021-9673(03)01261-5.

DOI:10.1016/s0021-9673(03)01261-5

PMID:14570324

Abstract

A method for detecting and quantifying bovine, ovine and caprine milk mixtures in milk and cheeses by means of reversed-phase high-performance liquid chromatography (RP-HPLC) of beta-lactoglobulins is described. Gradient elution was carried out with a flow rate of 0.5 ml/min and a temperature of 45 degrees C, using a mixture of two solvents: solvent A (0.1% TFA in water) and solvent B (0.09% TFA in 80% aqueous acetonitrile, v/v). The effluent was monitored at 215 nm. Under the conditions used different chromatographic patterns were obtained for bovine, ovine and caprine whey proteins. Each milk type presented different retention times for beta-lactoglobulin peaks. Binary mixtures of bovine and ovine or bovine and caprine raw milks containing 1, 2, 5, 10, 20, 30, 50, 75 and 95% (v/v) of bovine milk, as well as ovine and caprine milk mixtures containing 1, 2, 5, 10, 20, 30, 50, 75 and 95% (v/v) of ovine milk were used for cheese making. Cheeses were prepared and ripened, according to traditional methods. Milk mixtures, fresh and ripened cheeses were analyzed. A linear relationship was established between log 10 of beta-lactoglobulin peaks ratio (calculated as peak area values ratio) and log 10 of the relative percentage of bovine or ovine milk. The ratio between beta-lactoglobulin peaks was not affected by the degree of ripening. Thus, enabling the quantification of milk type percentage, with a detection limit of 2%. This technique allowed quantification of milk species within the concentration range of 5-95%. The method was successfully applied for authenticity evaluation and quantitative determination of ovine and caprine milk percentages of commercial protected denomination of origin (PDO) cheeses.

摘要

描述了一种通过β-乳球蛋白的反相高效液相色谱法（RP-HPLC）检测和定量牛奶及奶酪中牛、羊和山羊奶混合物的方法。采用两种溶剂的混合物进行梯度洗脱，流速为0.5 ml/min，温度为45℃，溶剂A（水中0.1%三氟乙酸）和溶剂B（80%乙腈水溶液中0.09%三氟乙酸，v/v）。在215 nm处监测流出物。在所使用的条件下，牛、羊和山羊乳清蛋白获得了不同的色谱图。每种牛奶类型的β-乳球蛋白峰具有不同的保留时间。含有1%、2%、5%、10%、20%、30%、50%、75%和95%（v/v）牛奶的牛和羊或牛和山羊生乳二元混合物，以及含有1%、2%、5%、10%、20%、30%、50%、75%和95%（v/v）羊奶的羊和山羊奶混合物用于制作奶酪。奶酪按照传统方法制备和成熟。对牛奶混合物、新鲜和成熟奶酪进行了分析。β-乳球蛋白峰面积比的对数10（以峰面积值比计算）与牛或羊奶相对百分比的对数10之间建立了线性关系。β-乳球蛋白峰之间的比例不受成熟程度的影响。因此，能够定量牛奶类型百分比，检测限为2%。该技术允许在5-95%的浓度范围内定量牛奶种类。该方法成功应用于商业受保护原产地名称（PDO）奶酪中羊和山羊奶百分比的真实性评估和定量测定。