Campbell R E, Boogers I A L A, Drake M A
Department of Food, Bioprocessing and Nutrition Sciences, Southeast Dairy Foods Research Center, North Carolina State University, Raleigh 27695.
DSM Biotechnology Center, Analysis Department, 6401 JH Delft, the Netherlands.
J Dairy Sci. 2014 Mar;97(3):1313-8. doi: 10.3168/jds.2013-7415. Epub 2013 Dec 28.
Norbixin is the primary carotenoid in annatto coloring, which imparts the desired orange color in Cheddar cheese. However, a portion of the colorant remains in the cheese whey and is undesirable; therefore, a bleaching step is often applied. Restrictions exist for norbixin concentrations in products destined for infant formula. As such, evaluation of norbixin concentrations in whey and whey ingredients is desirable. Current extraction methods are laborious and require solvents that are banned in many countries. The objective of this study was to develop a fast and inexpensive norbixin extraction and quantitation technique using approved solvents with similar sensitivity to current established methods. Instead of solvent extraction and column purification, acetonitrile was added directly to fluid wheys, retentates, and rehydrated whey protein concentrates. An isocratic mobile phase [70% acetonitrile and 30% water with 0.1% (wt/vol) formic acid] was used and, to increase sensitivity, a large volume (50 μL) was injected onto the column. The column used was a C18 column with a particle size of 2.6 μm and column length of 10 cm. The column inner diameter was 4.6mm and the pore size was 100 Ǻ. All of the previously described conditions allowed the run time to be only 4 min. The sample was sent through a photodiode array detector and quantified at 482 nm. Norbixin was quantified using external standard curves. The developed method had a >90% norbixin recovery in both milk and whey (9.39 μg/L-2.35 mg/L). The limit of detection of norbixin in fluid whey was 2.7 μg/kg and the limit of quantitation was 3.5 μg/kg, both of which are significantly lower than in previously described methods. The extracts were stable over 30 min at 21°C and stable over 24h at 4°C. Repeatability and precision of the method had relative standard deviations of less than 13%. The developed method provides time and cost savings for evaluation of norbixin concentration in whey and whey products.
胭脂树素是胭脂树色素中的主要类胡萝卜素，它能赋予切达干酪所需的橙色。然而，一部分色素会残留在奶酪乳清中，这是不理想的；因此，通常会进行漂白步骤。对于用于婴儿配方奶粉的产品中的胭脂树素浓度存在限制。因此，评估乳清和乳清成分中的胭脂树素浓度是很有必要的。目前的提取方法费力，且需要许多国家禁止使用的溶剂。本研究的目的是开发一种快速且廉价的胭脂树素提取和定量技术，使用经批准的溶剂，其灵敏度与当前已建立的方法相似。直接将乙腈添加到液态乳清、截留物和复水后的乳清蛋白浓缩物中，而不是进行溶剂萃取和柱纯化。使用等度流动相[70%乙腈和30%水，含0.1%（重量/体积）甲酸]，为提高灵敏度，将大体积（50μL）进样到柱上。所使用的柱是粒径为2.6μm、柱长为10cm的C18柱。柱内径为4.6mm，孔径为100Ǻ。上述所有条件使得运行时间仅为4分钟。样品通过光电二极管阵列检测器，并在482nm处进行定量。使用外标曲线对胭脂树素进行定量。所开发的方法在牛奶和乳清中（9.39μg/L - 2.35mg/L）的胭脂树素回收率均大于90%。液态乳清中胭脂树素的检测限为2.7μg/kg，定量限为3.5μg/kg，两者均显著低于先前描述的方法。提取物在21°C下30分钟内稳定，在4°C下24小时内稳定。该方法的重复性和精密度的相对标准偏差小于13%。所开发的方法为评估乳清和乳清产品中的胭脂树素浓度节省了时间和成本。
