Non-targeted and targeted analysis of collagen hydrolysates during the course of digestion and absorption

Abstract

Protein hydrolysates are an important part of the human diet. Often, they are prepared from milk, soy, or collagen. In the present study, four different collagen hydrolysates were tested, varying in the average molecular weight and the animal source. Three types of samples, the dissolved start products, in vitro generated dialysates (containing the digested components that are potentially available for small intestinal absorption), and human serum collected after product ingestion, were analyzed using LC-MS to compare the state of the hydrolysates before and after absorption, i.e., uptake into the blood. It was found that the composition of the collagen hydrolysates prior to and after ingestion was highly complex and dynamic, which made it challenging to predefine a strategy for a targeted analysis. Therefore, we implemented a new analytical approach to first map hydrolysate data sets by performing non-targeted LC-MS analysis followed by non-targeted and targeted data analysis. It was shown that the insight gained by following such a top down (data) analytical workflow could be crucial for defining a suitable targeted setup and considering data trends beyond the defined targets. After having defined and performed a limited targeted analysis, it was found that, in our experimental setup, Hyp-Gly and especially Pro-Hyp contributed significantly as carrier to the total Hyp increase in blood after ingestion of collagen hydrolysate.

Skin collagen through the lifestages: importance for skin health and beauty

“…As collagen is uniquely rich in glycine, proline (and hydroxyproline, which is derived by posttranslational modification during collagen synthesis), this represents an enriched supply of the specific amino acids required to make new collagen fibrils. Secondly, unique oligopeptide sequences, especially dipeptides containing hydroxyproline, are known to stimulate fibroblasts via receptor-mediated activation pathways to induce new collagen fibre synthesis [95] . Although present at lower levels than amino acids, the peptides can stimulate fibroblast receptors and are thus biologically potent even at lower absolute concentrations.…”

“…Even if we account for an enrichment of glycine, proline and hydroxyproline species, the number of peptides which can potentially stimulate fibroblasts to synthesis new collagens is too large to test in vivo. Research has shown that significant amounts of the di-and tri-peptide species, Pro-Hyp, Ala-Hyp, Ala-Hyp-Gly, Pro-Hyp-Gly, Leu-Hyp, Ile-Hyp and Phe-Hyp were measurable in human blood following oral ingestion of different collagen hydrolysates [95][96][97][98][99][100] . Some of these di-or tri-peptides have been shown to stimulate fibroblasts in vitro [101,102] .…”

Reliable and generic liquid chromatography/mass spectrometry quantification of short peptides using a stable‐isotope‐labeled labeling agent

“…To explore the dynamic range, spikes were prepared in solvent and in simulated gastrointestinal fluid 20 using GP p and P p at the concentrations 80 μg/mL, 40 μg/mL, 4 μg/mL, 400 ng/mL, 40 ng/mL, 4 ng/mL and 0 ng/mL and these samples were prepared and analyzed using the same conditions as the FBS (spikes), except that 150 μL of MQ was added prior to LC/MS analysis. Linear regression with 1/x 2 weighting was used to calculate the calibration curve equations.…”

“…Collagen hydrolysates are important nutraceutical products as it has been reported that they can support bone, joint and skin health 16–19 . Previously we used an approach with matrix‐matched external calibration 20 and labeling with 6‐aminoquinolyl‐ N ‐hydroxysuccinimidyl carbamate (AQC) 21–25 to quantify peptides of interest. AQC derivatization can also be used to analyze amino acids and peptides that do not originate from collagen 21–25 and generally improves the ionization efficiency and chromatographic performance in reversed‐phase LC of amino acids and short peptides 22,25 .…”

Potentiating cutaneous wound healing in young and aged skin with nutraceutical collagen peptides

“…Cells were seeded on to 96-well plates or coverslips, which were either uncoated or coated with 1 mg/mL bovine or porcine collagen peptide prior to treatment for 2 h in the presence/absence of 7.5 µg/mL mitomycin C (Sigma-Aldrich) and incubated at 37°C for 72 h. 5 mL fruit juice (Ribena; Suntory, Osaka, Japan) diluted in 250 mL water following an overnight fast. Venepuncture blood samples were obtained at 0, 2, 8 and 24 h post-ingestion, with serum and plasma samples subsequently stored at À80°C prior to quantification of total Hyp by ultra-performance liquid chromatography tandem mass spectrometry, as previously described 21 (Data S1).…”