Free Amino Acid Analysis of Protein Hydrolysates and Peptones
Compare hydrolysate, peptone and yeast extract lots on the part that is already free amino acid: 19 amino acids plus ammonia, an optional extended panel, and powders prepared in-house.
A hydrolysate is part free amino acids and part peptides, and the split shifts with raw material and process. We measure the free fraction. That is the right number for comparing lots and suppliers on free amino acids, and it is not a total composition.
What we measure in protein hydrolysates and peptones
Protein hydrolysates are mixtures of free amino acids, peptides and carbohydrates, and can also contain vitamins and lipids. Their make-up depends on the protease, hydrolysis time and temperature, and the raw material 1. Our free amino acid profile (AA-1) quantifies 19 amino acids plus ammonia by AQC derivatization, reversed-phase HPLC and UV detection at 260 nm, in µM and mg/L. Because nothing is hydrolyzed, glutamine, asparagine and tryptophan are reported as themselves. Acid hydrolysis for a total analysis destroys tryptophan and converts asparagine and glutamine into aspartic and glutamic acid 2.
| Service | What it adds for hydrolysates |
|---|---|
| AA-1 | 19 free amino acids plus ammonia: the free fraction as delivered |
| AA-2 (+$25/sample) | GABA, ornithine, citrulline, taurine, hydroxyproline. In soy hydrolysate work reviewed by Ho et al., lots high in ornithine and citrulline correlated with higher antibody titer in CHO culture. Lots high in adenosine and arginine correlated with lower titer 1. |
| PREP-1 (+$30/sample) | Powders weighed and dissolved in-house |
The free share varies more than a product name suggests. Degree of hydrolysis (DH) is the proportion of peptide bonds broken 1, but it does not map neatly onto free amino acids. Three whey hydrolysates at DH 48%, 27% and 23% contained an estimated 21%, 2% and 6% free amino acids 3. Enzymatic hydrolysis of trout viscera reached DH 83.8% and released 71.3% of the total protein as free amino acids; acid autolysis reached 75.8% and 52.5% 4. For yeast extracts, reported free shares range from 11–15% after mechanical cell disruption to 77.5% after 48 hours of autolysis 5.
Lots differ too. Hydrolysates are undefined, which leads to batch-to-batch quality issues in cell culture 1. One NMR survey covered 27 samples (2–4 lots each) of eight plant- and yeast-based hydrolysates from one manufacturer. Lot-to-lot variation was driven by a small set of compounds, including lysine in one yeast extract 6. Yeast extracts from different suppliers, and lots from one supplier, changed recombinant protein production by E. coli 7 and alginate production by A. vinelandii up to twofold 8. See also fermentation and cell culture media.
Questions this answers
- How much of this hydrolysate is already free amino acid, and which amino acids make it up?
- Is the new lot of peptone, yeast extract or soy hydrolysate consistent with the lot that worked?
- Which of two suppliers' casein or soy hydrolysates matches the free amino acid profile we specify?
- Did a change of enzyme, hydrolysis time or temperature shift the free fraction?
- Does the product carry free glutamine, asparagine and tryptophan that a total analysis cannot report?
- Which free amino acids move most between lots, so we know what to watch?
Caveats for this matrix
Free is not total
A free profile leaves out every residue still bound in peptides. Peptides can exceed 50% of yeast extract mass 9. In the NMR survey above, the free small molecules the method could assign averaged about 17% of product mass, and 43% for one yeast extract 6. Total composition after acid hydrolysis is limited to 17 amino acids 2, and we do not offer it yet. Compare free results with free results, not with a total-composition specification; see free vs total amino acids.
High concentrations need dilution
A product that is largely free amino acid 4 can sit far above the method's working range. We expect 5 µM–5 mM per analyte and dilute concentrated samples in-house to bring them into range. Dilution also protects the 4–6-fold molar excess of AQC that complete derivatization needs 10. Results below the quantitation limit are flagged, not reported as numbers.
Powders and acidic products
We weigh and dissolve powders in-house under PREP-1 (sample preparation). AQC derivatization needs the reaction buffered at roughly pH 8.2–10.1, and below 8.2 it is incomplete 10. That matters for products made or held under acidic conditions, such as acid autolysis 4. We check and adjust reaction pH, and the first time we see a matrix type we run a spike-recovery check (PREP-2).
Flavor and function sit partly in the peptides
In wheat gluten hydrolysates, bitterness rose as DH went from 4% to 20%. The fraction below 180 Da, mainly free amino acids, was not bitter; peptides of 500–1,000 Da were the most bitter 11. Yeast extract lots can also change culture performance in ways one study did not trace to specific components 7. A free profile is one input to supplier qualification, not the whole of it. Our application note on the free fraction reviews the published evidence.
Application notes
New publications on protein hydrolysates & peptones analysis are reviewed monthly; relevant findings and what they change for sampling or interpretation are added to this page.
Key references
- Applications and analysis of hydrolysates in animal cell culture — Ho YY, Lu HK, Lim ZFS, Lim HW, Ho YS, Ng SK. Bioresources and Bioprocessing, 2021.
- 〈1052〉 Biotechnology-Derived Articles—Amino Acid Analysis — United States Pharmacopeia, harmonized general chapter (official May 1, 2018).
- Effect of degree of hydrolysis of whey protein on in vivo plasma amino acid appearance in humans — Farup J, Rahbek SK, Storm AC, Klitgaard S, Jørgensen H, Bibby BM, Serena A, Vissing K. SpringerPlus, 2016.
- Comparison of amino acid release between enzymatic hydrolysis and acid autolysis of rainbow trout viscera — Domínguez H, Iñarra B, Labidi J, Mendiola D, Bald C. Heliyon, 2024 (Zenodo record).
- The complexity of yeast extracts and its consequences on the utility in brewing: A review — Jacob FF, Methner FJ, Michel M, Zarnkow M, Hutzler M. BrewingScience, 2019.
- NMR metabolomics of plant and yeast-based hydrolysates for cell culture media applications — A comprehensive assessment — Combe M, Isaac KS, Potter G, Sokolenko S. Current Research in Food Science, 2024.
- Phenotyping the quality of complex medium components by simple online-monitored shake flask experiments — Diederichs S, Korona A, Staaden A, Kroutil W, Honda K, Ohtake H, Büchs J. Microbial Cell Factories, 2014.
- Yeast extracts from different manufacturers and supplementation of amino acids and micro elements reveal a remarkable impact on alginate production by A. vinelandii ATCC9046 — Sparviero S, Dicke MD, Rosch TM, Castillo T, Salgado-Lugo H, Galindo E, Peña C, Büchs J. Microbial Cell Factories, 2023.
- Insights Into the Complexity of Yeast Extract Peptides and Their Utilization by Streptococcus thermophilus — Proust L, Sourabié A, Pedersen M, Besançon I, Haudebourg E, Monnet V, Juillard V. Frontiers in Microbiology, 2019.
- Derivatization of Amino Acids Using Waters AccQ•Tag Chemistry — Waters Corporation, Comprehensive Guide to Hydrolysis and Analysis of Amino Acids (primer), n.d.
- Characteristics of the enzyme-induced release of bitter peptides from wheat gluten hydrolysates — Sun X, Zheng J, Liu B, Huang Z, Chen F. Frontiers in Nutrition, 2022.
OpenChemWorks Laboratory · Reviewed by the laboratory director, PhD chemist · Updated September 23, 2026
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