Oral collagen supplements are typically derived from bones, cartilage, and tendons of bovine, poultry, porcine, and marine sources (Kržišnik et al., 2024). Collagen has gained significant attention for its potential benefits on aging skin, including improved skin appearance, hydration, elasticity, firmness, wrinkles reduction, and wound healing acceleration, among other benefits (Kržišnik et al., 2024). Additionally, collagen supplements such as collagen hydrolysates have been used to support joint health, particularly in cases of arthritis and osteoarthritis (Lopez-Morales et al., 2019). Undenatured collagen has been recognized for its suppressive effects on degradative autoimmune reactions in the joints by promoting regulatory T cell (Tregs); this targeted immunomodulatory response is considered favorable in the management of collagen-induced arthritis (Park et al., 2009). As a widely popular and multifunctional product, it is essential to ensure collagen supplements’ quality, safety, and efficacy (Kržišnik et al., 2024). This Collagen Science Update reports on novel analytical methods for assessing collagen sources and supplements, quality control measures, and more.  

Analytical testing of collagen summary 

One of the most widely used approaches to analyze collagen is the Association of Official Analytical Chemists (AOAC) Method 990.26, which combines acid hydrolysis and colorimetric hydroxyproline assay, as hydroxyproline is the predominant amino acid in most bovine and porcine collagen sources (GME, 2023). Although alternative techniques such as high-performance liquid chromatography (HPLC) have also been utilized for collagen quantification in meat products, they have limitations, including high operational costs (da Silva et al., 2015). 

An additional methodology employed for the quantitation of collagen in its active (i.e. undenatured) form is the Enzyme-Linked Immunosorbent Assay (ELISA). This technique allows for the determination of collagen that is available for immune-mediated responses to affected areas of the body, such as the joints. ELISA has some limitations, such as its dependence on uniform, targeted sample extraction processes, variability in generating a live antibody-antigen complex for detection, and a lack of speed regarding testing throughput (Park et al., 2009). However, this methodology exhibits analytical superiority in terms of accuracy, specificity, and sensitivity. Various ELISA kits are also commercially available for the quantification of individual collagen subtypes; a useful analytical tool in assessing material composition and determining viability of subtype-specific treatment capabilities (Park et al., 2009). 

According to the United States Pharmacopeia (USP) Gelatin Monograph, gelatin is identified using infrared spectroscopy, while the presence of peptides is confirmed through a colorimetric assay (USP, 2019). Additional quality tests include water conductivity, sulfur dioxide and peroxide content analysis, loss on drying, microbial enumeration tests, determination of gel strength (bloom value) of the sample, testing for specified microorganisms (Salmonella sp. and Escherichia coli), and analysis for metals including iron, chromium, and zinc (USP, 2019). 

The Gelatine Manufactures of Europe (GME), in their Collagen Peptides Monograph (2023), outline several standardized test methods. To identify collagen peptides, they include the Biuret method for protein determination, assessment of gelling versus non-gelling characteristics, and hydroxyproline determination. Tests for chemical characteristics include loss on drying ash, nitrogen, and a distillation method to test sulfur dioxide. Atomic spectrometry is used to test metals such as arsenic, copper, iron, lead, and zinc (GME, 2023). The monograph also describes tests for physical properties including viscosity, colour, clarity, pH, and conductivity, as well as microbiological characteristics such as total aerobic microbial count (TAMC), Salmonella, Escherichia coli, and sulphite-reducing anaerobic spores. Similarly, GME’s Gelatin Monograph (2023) outlines testing methods for gelatin that closely align with those described in the Collagen Peptides Monograph.  

A comprehensive analytical approach for quality control of collagen in food supplements 

Collagen is attracting widespread attention in the food, pharmaceutical, nutraceutical, and cosmetic industries due to its many health benefits, especially its ability to enhance the appearance of skin. However, as its use expands, rigorous quality control measures are essential to ensure the safety and efficacy of collagen. Typically, oral collagen supplements are derived from bovine, porcine, marine, or poultry sources, which may introduce the risk of toxins and contaminants during extraction. Furthermore, certain types of collagen carry a risk of disease transmission; for example, marine collagen may trigger allergic reactions in individuals with seafood allergies. Currently, the quantitative determination of collagen primarily relies on time-consuming and non-selective spectroscopic methods or on complex chromatographic methods involving derivatization or mass spectrometric detection. As such, there is a need for new, more efficient and selective methods for collagen testing. This study investigated improvements to current spectroscopic techniques, particularly in specificity, repeatability, stability, and simplification in sample preparation, using chromatographic methods. The study tested 1) HPLC to quantify hydroxyproline (Hyp) (HPLC-dHyp), 2) bacterial collagenase-enzymatic hydrolysis forming N-terminal glycine-containing peptides (NGPs) (HPLC-dNGP), 3) determination of collagens through the underivatized tripeptide Gly-Pro-Hyp (HPLC-GPH), and 4) analyse collagen hydrolysates in untreated samples of food supplements. Also, other methods such as fluorescence detection using o-phthalaldehyde (FLD-OPA) and ultraviolet detection with bicinchoninic acid (UV-BCA) were tested. Overall, these new analytical methods demonstrated superiority in terms of specificity, repeatability, sample stability, and simplification in the sample preparation relative to traditionally used methods for collagen assessment. New analytical methods and quality control measures are warranted to assess collagen in food supplements.  

Access to the study: https://www.mdpi.com/1660-3397/22/10/435  

Reference: Kržišnik, N., Kurent, E., & Roškar, R. (2024). A Comprehensive Analytical Approach for Quality Control of Collagen in Food Supplements. Marine Drugs, 22(10), 435. https://doi.org/10.3390/md22100435  

Determination of peptide profile consistency and safety of collagen hydrolysates as quality attributes 

The present study proposes an analytical approach to determine the composition and peptide profile, as a quality attribute, of a collagen product called Colagenart® containing collagen hydrolysates. Collagen hydrolysates are complex mixtures of low-molecular-weight peptides derived from the enzymatic hydrolysis of collagen, characterized by intrinsic batch-to-batch heterogeneity. However, their quality is dependent on the reproducibility of their mass distribution pattern, which 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 results demonstrated batch-to-batch reproducibility, supporting the utility of this approach in establishing consistency of the quality attributes of collagen hydrolysates. Furthermore, the study addressed the product’s safety by exposing and measuring the viability of two cell lines, CaCo-2 and HepG2, with no negative effects observed.   

Access to the study: https://ift.onlinelibrary.wiley.com/doi/epdf/10.1111/1750-3841.14466  

Reference: López-Morales, C. A., Vázquez-Leyva, S., Vallejo-Castillo, L., Carballo-Uicab, G., Muñoz-García, L., Herbert-Pucheta, J. E., Zepeda-Vallejo, L. G., Velasco-Velázquez, M., Pavón, L., Pérez-Tapia, S. M., & Medina-Rivero, E. (2019). Determination of Peptide Profile Consistency and Safety of Collagen Hydrolysates as Quality Attributes. Journal of Food Science, 84(3), 430–439. https://doi.org/10.1111/1750-3841.14466  

Picrosirius red staining: Revisiting its application to the qualitative and quantitative assessment of collagen type I and type III in tendon 

Picrosirius red (PSR) staining, viewed under polarized light microscopy, is used as the standard method to evaluate collagen fibers organization and to distinguish collagen types I and III in tissue samples. However, challenges such as historical misconceptions and technical factors hinder obtaining accurate analyses and interpretations from PSR images. As such, this study aimed to clarify whether collagen types I and III can be reliably distinguished using PSR staining in rat Achilles tendons, using double immunohistochemistry as the positive control. The findings indicated that PSR staining viewed under polarized light microscopy is suitable for qualitative and quantitative assessment of total collagen but is not reliable for distinguishing between collagen types. Immunohistochemistry was superior to PSR staining for collagen type III detection. Lastly, the study compared formalin and Bouin solutions as fixatives, where formalin-fixed tendons provided higher histological detail quality with both staining techniques- hematoxylin-eosin and immunostaining. Therefore, PSR can be used with double immunohistochemical labeling when the precise distinction of collagen types I and III is needed. PSR staining is a low-cost, effective tool to examine collagen fiber content, organization, orientation, and possibly total collagen density and quantification. Nonetheless, the correct and standardized use of PSR staining is necessary to ensure reliable and reproducible results.   

Access to the study: https://pubmed.ncbi.nlm.nih.gov/34549650/  

Reference: López De Padilla, C. M., Coenen, M. J., Tovar, A., De la Vega, R. E., Evans, C. H., & Müller, S. A. (2021). Picrosirius Red Staining: Revisiting Its Application to the Qualitative and Quantitative Assessment of Collagen Type I and Type III in Tendon. The Journal of Histochemistry and Cytochemistry : Official Journal of the Histochemistry Society, 69(10), 633–643. https://doi.org/10.1369/00221554211046777  

Quality parameters of collagen containing raw materials and courses of their use 

This study addresses the quality parameters of collagen-containing minced meat (CMM) and collagen-containing gel (CCG), both derived from the processing of hen legs. It evaluates the effect of these characteristics on finished meat products and provides recommendations for their use in meat product technology. CMM and CCG differ in chemical composition and collagen content despite being obtained from collagen-containing raw materials. Quality parameter evaluation of CMM and CCG showed that both are promising raw materials for the meat industry, as they contain significant amounts of total protein and collagen and exhibit favorable sensorial characteristics. The addition of 20% CMM or 20% CCG to minced meat influences the rheological parameters of the finished products, particularly the final shear stress and elasticity, which reported an increase in the samples with CMM compared to control. Regarding samples with CCG, only the elasticity index was increased. These results highlight the importance of establishing differential approaches for the use of collagen-like raw materials in meat products, considering these products’ individual peculiarities and characteristics.  

Access to the study: https://ibn.idsi.md/vizualizare_articol/117762  

Reference: Voytsekhivska, L., Franko, O., Okhrimenko, Y., Verbytskyi, S., & Gavrilenko, A. (2020). Quality parameters of collagen containing raw materials and courses of their use. Journal of Engineering Science, 27(4), 183-190. https://doi.org/10.5281/zenodo.4288320  

Future trends of analytical testing of collagen  

 Overall, advanced analytical techniques, such as HPLC-based methods, may drive future trends in collagen testing due to their sensitivity and precision. However, their widespread adoption is limited by high operational costs (da Silva et al., 2015). Additionally, there is growing interest in developing methods that are less time-consuming than conventional approached, such as the hydroxyproline assay (post-hydrolysis) (Kržišnik et al., 2024). Novel methods to identify the animal source of collagen would allow for traceability and enhanced quality control. 

Bottom Line 

This report highlights significant advancements in collagen quality assessment and application. Novel HPLC-based methods and related analytical techniques have improved specificity, repeatability, and ease of testing for collagen supplements. Studies confirm the importance of testing for batch-to-batch consistency and safety of collagen hydrolysates to support their continued use. While Picrosirius Red staining remains a cost-effective technique for analyzing collagen fibers, it lacks precision in differentiating collagen types, which may be enhanced with the use of immunohistochemistry.  Additionally, collagen-containing meat by-products show potential as high-quality raw materials in the food industry, impacting texture and nutritional value. Altogether, these findings support the growing use of collagen across nutraceutical and food applications, while emphasizing the need for rigorous quality control standards.  

References 

AOAC Official Method 990.26. Hydroxyproline in Meat and Meat Products: Colorimetric Method’, in Dr. George W Latimer, Jr. (ed.), Official Methods of Analysis of AOAC INTERNATIONAL, 22nd Edition (New York, 2023; online edn, AOAC Publications, 4 Jan. 2023), https://doi.org/10.1093/9780197610145.003.3457 

da Silva, C. M., Spinelli, E., & Rodrigues, S. V. (2015). Fast and sensitive collagen quantification by alkaline hydrolysis/hydroxyproline assay. Food chemistry, 173, 619–623. https://doi.org/10.1016/j.foodchem.2014.10.073 

EFSA Panel on Dietetic Products, Nutrition and Allergies; Scientific Opinion on the substantiation of a health claim related to collagen hydrolysate and maintenance of joints pursuant to Article 13(5) of Regulation (EC) No 1924/2006. EFSA Journal 2011; 9(7):2291. [11 pp.]. doi:10.2903/j.efsa.2011.2291. 

Gelatine Manufacturers of Europe. (August 2023). Standardised methods for the testing of edible collagen peptides (Collagen Peptides Monograph). https://www.gelatine.org/fileadmin/user_upload/gme_content/GME_Statements/GME_CP_Monograph__August_2023.pdf 

Gelatin Manufactures of Europe. (August 2023). Standardised Methods for the Testing of Edible Gelatine (Gelatine Monograph). https://www.gelatine.org/fileadmin/user_upload/gme_content/GME_Statements/GME_Gelatine_Monograph_August_2023.pdf 

León-López A, Morales-Peñaloza A, Martínez-Juárez VM, Vargas-Torres A, Zeugolis DI, Aguirre-Álvarez G. Hydrolyzed Collagen-Sources and Applications. Molecules. 2019 Nov 7;24(22):4031. doi: 10.3390/molecules24224031. PMID: 31703345; PMCID: PMC6891674. 

Park, K. S., Park, M. J., Cho, M. L., Kwok, S. K., Ju, J. H., Ko, H. J., … Kim, H. Y. (2009). Type II collagen oral tolerance; mechanism and role in collagen-induced arthritis and rheumatoid arthritis. Modern Rheumatology, 19(6), 581–589. https://doi.org/10.3109/s10165-009-0210-0 

The United States Pharmacopeia. (April 2019). Stage 4 Harmonization. https://www.usp.org/sites/default/files/usp/document/harmonization/excipients/harmonization-april-2019-m34770.pdf