Determination of Peptide Profile Consistency and Safety of Collagen Hydrolysates as Quality Attributes

Abstract

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 [ 1 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. [ 1 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.

Enzymatic Hydrolysis of a Collagen Hydrolysate Enhances Postprandial Absorption Rate—A Randomized Controlled Trial

“…Baar et al [23] showed that AAs present in high amounts in collagen (glycine, proline, lysine, hydroxyproline and hydroxylysine) combined with vitamin C could improve collagen synthesis in a tissue-engineered ligament model. Similar findings were reported by de Paz-Lugo et al [20], who found that glycine and proline enhanced type II collagen synthesis in bovine articular chondrocytes, suggesting that increased consumption of these AAs could be beneficial for the regeneration of the articular cartilage matrix. Oesser et al [24] investigated the absorption of gelatin hydrolysate and its subsequent tissue distribution in mice.…”

“…Plasma concentrations of glycine and proline peaked 60 and 90 min after intake of EHC and NC, respectively. Previous studies have reported similar findings with a peak in hydroxyproline after 120 min and a subsequent decrease to half the maximum after four hours following consumption of hydrolyzed collagen [20,27].…”

“…Accordingly, the products’ content of heavy metals has been analyzed and the values are considered safe. We have not conducted any further testing of the products in relation to safety, but a recent study with a similar collagen product revealed that the product did not elicit any toxic effects in an in vitro model [20].…”

Collagen Hydrolysates for Skin Protection: Oral Administration and Topical Formulation

“…Human consumption of dietary supplements such as hydrolyzed collagen has been deemed to be safe. Lopez-Morales et al [103] conducted a detailed experimental work to characterize HC with different techniques such as ultraviolet detection (SEC-UV), reverse phase chromatography coupled to electrospray ionization ion mobility quadrupole time-of-flight spectrometer (RP-HPLCESI-IMS-QTOF), and shaped-pulse water-suppression one (1D)-and two-dimensional (2D) proton nuclear magnetic resonance spectra. Safety and toxicity of HC were assessed in vitro by using CaCo-2 and HepG2 cell lines.…”

Ion Mobility Spectrometry in Food Analysis: Principles, Current Applications and Future Trends

“…Nowadays, IMS applications in food composition cover a wide variety of substances (e.g., lipids, peptides, phenolic compounds, terpenes, etc.) [44,45,46,47], including allergens [48], and samples (e.g., flours, olive oil, mushrooms, etc.) [48,49,50].…”