Unidad de Desarrollo e Investigación en Bioprocesos (UDIBI), Escuela Nacional de Ciencias Biológicas, Insto. Politécnico Nacional, Ciudad de México, 11340, México.
Depto. de Farmacología, Cinvestav IPN. Ciudad de México 07360, México.
J Food Sci. 2019 Mar;84(3):430-439. doi: 10.1111/1750-3841.14466. Epub 2019 Feb 15.
Collagen hydrolysates are dietary supplements used for nutritional and medical purposes. They are complex mixtures of low-molecular-weight peptides obtained from the enzymatic hydrolysis of collagen, which provide intrinsic batch-to-batch heterogeneity. In consequence, the quality of these products, which is related to the reproducibility of their mass distribution pattern, should be addressed. Here, we propose an analytical approach to determine the peptide pattern as a quality attribute of Colagenart®, a product containing collagen hydrolysate. In addition, we evaluated the safety by measuring the viability of two cell lines exposed to the product. The consistency of peptide distribution was determined using Size Exclusion Chromatography (SEC), Mass Spectrometry coupled to a reversed phase UPLC system (MS-RP-UPLC), and Shaped-pulse off-resonance water-presaturation proton nuclear magnetic resonance spectrometry [ Hwater_presat NMR]. The mass distribution pattern determined by SEC was in a range from 1.35 to 17 kDa, and from 2 to 14 kDa by MS-RP-UPLC. [ Hwater_presat NMR] showed the detailed spin-systems of the collagen hydrolysates components by global assignment of backbone Hα and NH, as well as side-chain proton resonances. Additionally, short-range intraresidue connectivity pathways of identified spin-regions were obtained by a 2D homonuclear shift correlation Shaped-pulse solvent suppression COSY scheme. Safety analysis of Colagenart® was evaluated in CaCo-2 and HepG2 cells at 2.5 and 25 μg/mL and no negative effects were observed. The results demonstrated batch-to-batch reproducibility, which evinces the utility of this approach to establish the consistency of the quality attributes of collagen hydrolysates. PRACTICAL APPLICATION: We propose state-of-the art analytical methodologies (SEC, MS, and NMR) to evaluate peptide profile and composition of collagen hydrolysates as quality attributes. These methodologies are suitable to be implemented for quality control purposes.
胶原蛋白水解物是一种用于营养和医疗目的的膳食补充剂。它们是胶原蛋白酶解得到的低分子量肽的复杂混合物，具有内在的批次间异质性。因此，应该解决这些产品的质量问题，这与其质量分布模式的重现性有关。在这里，我们提出了一种分析方法来确定 Colagenart®（一种含有胶原蛋白水解物的产品）的肽图谱作为质量属性。此外，我们通过测量两种细胞系暴露于该产品的活力来评估安全性。使用尺寸排阻色谱（SEC）、与反相 UPLC 系统（MS-RP-UPLC）偶联的质谱以及形状脉冲去偶水预饱和质子核磁共振波谱 [ Hwater_presat NMR] 来确定肽分布的一致性。SEC 确定的分子量分布范围为 1.35 至 17 kDa，而 MS-RP-UPLC 确定的分子量分布范围为 2 至 14 kDa。[ Hwater_presat NMR] 通过对胶原蛋白水解物成分的骨架 Hα 和 NH 以及侧链质子共振的全局分配，显示了详细的自旋系统。此外，通过二维同核位移相关 Shaped-pulse 溶剂抑制 COSY 方案获得了鉴定的自旋区域的短程残基内连接途径。在 2.5 和 25 μg/mL 的浓度下，在 CaCo-2 和 HepG2 细胞中评估了 Colagenart®的安全性，未观察到负面作用。结果表明批次间具有重现性，证明了该方法在建立胶原蛋白水解物质量属性一致性方面的实用性。实际应用：我们提出了最先进的分析方法（SEC、MS 和 NMR）来评估胶原蛋白水解物的肽图谱和组成作为质量属性。这些方法适用于质量控制目的。
