Hydrolysates are proteins digested into smaller fragments, peptides, and its sole building blocks, the amino acids. So a hydrolysate is a blend of different peptides and different peptide lengths. In the hydrolysis process, we start with the intact protein, for instance whey or casein, which is digested by enzymes into smaller fragments. The hydrolysis process influences the peptide size. Depending on the level of hydrolysis, we are able to obtain mildly hydrolysed, partially hydrolysed or extensively hydrolysed proteins. Moreover, the peptides can be filtered, in order to obtain a mixture with very small peptides and no larger fragments.
Process steps of typical filtered hydrolysate production process based on enzymes
Raw material selection
The production of protein hydrolysates starts with the selection of the protein substrate and enzymes. FrieslandCampina Domo procures high quality raw materials from its suppliers, which pass through several quality checks before they are used in the production process.
The protein substrate is solubilised in water at a given temperature. pH of the protein solution is adjusted to achieve optimal conditions for hydrolysis.
The enzyme is added to the protein solution and hydrolysis starts. The temperature and the pH are continuously monitored and controlled during the process of hydrolysis.
Once the appropriate degree of hydrolysis (% DH) is achieved, the solution is heated to a target temperature for a period of time.
Filtration and concentration
The filtration procedures facilitate the removal of any undesirable matter and guarantee a clear hydrolysed protein solution.
Ultrafiltration reduces the endotoxins in the hydrolysed protein solution at a well-controlled feed rate and temperature.
Finally, the protein solution is concentrated and spray dried to yield a high quality consistent protein hydrolysate.
Packaging & Storage
Protein hydrolysates are hygroscopic. Therefore, storage should be done in a sealed air-tight container.
Parameters to describe the hydrolysis characteristics of protein hydrolysates
FrieslandCampina Domo uses a number of different parameters to describe the hydrolysis characteristics of protein hydrolysates. The technical overview below gives an explanation on these parameters and the techniques which are used to characterise our products.
Proteins are long chains of amino acids linked through peptide bonds. Upon (enzymatic) hydrolysis these peptide bonds are broken, resulting in a peptide mixture ranging from small peptides (a few amino acids linked together) to free amino acids (see figure 1). Depending on the type of enzyme(s) used and processing conditions chosen, different patterns of peptides result. This determines to a large extent the nutritional and technological features of protein hydrolysates.
Figure 1 – Hydrolysis of peptide bonds in a protein.
Amino Nitrogen (AN) and Total Nitrogen (TN)
Upon enzymatic hydrolysis of the peptide bonds in a protein, new amino groups are formed, one amino group for each broken peptide bond. The amount of newly formed amino groups causes a linear increase in Amino Nitrogen (AN). The AN content is usually determined by formol titration1, although other methods are available. Amino Nitrogen (AN) represents the amount of all amino groups in a hydrolysate or protein, which include those in free amino acids, the N-termini of peptides and proteins and the side chain amino groups in lysine. Next to AN, also the total nitrogen content (TN) of hydrolysates is measured. TN is quantitatively determined by the Kjeldahl method2. TN includes all nitrogen in the product, including non-protein nitrogen, and nitrogen incorporated in amino acid side chains.
Calculation of the degree of hydrolysis
The degree of hydrolysis (DH) is defined as the percentage of the number of peptide bonds in a protein which have been cleaved during hydrolysis (Adler-Nissen3, see formula 1) where htot is the number of peptide bonds per protein equivalent, and h is the number of hydrolysed bonds. htot is dependent on the amino acid composition of the raw material.
To calculate DH, it is necessary to have values for h and htot. Adler-Nissen has proposed the following formula for this: (see formula 2) where AN1 is the amino nitrogen content of the protein substrate before hydrolysis (mg/g protein), AN2 the amino nitrogen content of the protein product after hydrolysis (mg/g protein) and Npb the nitrogen content of the peptide bonds in the protein substrate (mg/g protein).
Adler-Nissen calculated Npb from an amino acid analysis, by summing up the moles of each individual amino acid per gram, resulting in a value of 114.8 mg/g protein for casein and 123.2 mg/g protein for whey3. However, this calculation can be improved when taking into account the proportion of the individual proteins in whey protein (α-lactalbumin, β-lactoglobulin, bovine serum albumin and CMP) or casein (α-casein, β-casein and κ-casein).
Domo calculated this Npb, both for whey and casein, using the proportion of the individual proteins and amino acid sequence data obtained from the SWISS-PROT data base, resulting in a value of 122.2 mg/g protein for casein and 126.0 mg/g protein for cheese whey.
Since the protein content in substrate and product may differ (hydrolysates have in most cases a lower protein content than their substrates), FrieslandCampina Domo adapted the formula by dividing the AN values by their corresponding TN. Doing so, also Npb has to be divided by TN of the substrate, resulting in the following formula for DH (see formula 3) where (AN/TN)product is the AN/TN ratio of the hydrolysate, (AN/TN)substrate is the AN/TN ratio of the raw material and (Npb/TN)substrate is the protein specific ratio of nitrogen present in the peptide bonds and total nitrogen content of the raw material.
Amino Nitrogen (AN) and Total Nitrogen (TN) are most frequently analysed by chemical methods (formol titration and Kjeldal respectively). For substrate proteins with limited solubility, calculating AN and TN based on sequence data and the proportion of the individual proteins in the substrate may be the more reliable option. Since the AN analysis (formol titration) measures also the N in the side chain of lysine, the theoretical calculation of AN of the raw material needs to be based on both the number of N-termini and amount of lysine in the raw material.
Based on these protein specific constants, the DH formula for casein and whey protein can be simplified (see formula 4 and 5). For proteins with a more complex composition (e.g. soy, wheat), AN, TN and number of peptide bonds can be estimated based on the amino acid composition.
Molecular weight distribution (MWD)
The molecular weight distribution (MWD) profile reflects the functional and nutritional properties of the protein hydrolysates. This molecular weight distribution profile is commonly measured using Gel Permeation Chromatography (GPC). Using this method, peptides are separated according to their size and shape, which is related to their molecular weight. In general, larger peptides are eluted first, smaller ones come later. The type of reagents, column and standards used will affect the results obtained. It must be emphasized that MWD profiles of different hydrolysates must always be measured under one set of conditions; results obtained from different methods cannot be compared directly.
In a method applied by FrieslandCampina Domo, a water/ acetonitrile/trifluoroacetic acid (69.9/30/0.1) eluent on a Progel TSK-G2000SWXL column with UV detection at 214nm is used to measure the MWD profile of protein hydrolysates. An example of a MWD profile obtained by this method is given in figure 2. Because larger peptides leave the column first, the higher molecular weights can be found to the left of the scale and the lower weights at the right.
Figure 2 – Molecular Weight Distribution profile (MWD) of a protein hydrolysate
The results are usually represented in a table as % distribution of the molecular weight classes MW <500; 500-1,000; 1,000-2,000; 2,000-5,000; 5,000-10,000 and >10,000 Dalton (see figure 2). Free amino acids are only partially picked up by this technique: only those amino acids that have an absorption at 214nm (e.g. tyrosine and tryptophan) are detected. So the class MW <500 Dalton generally does not include the free amino acids.
Another technique to separate proteins according to their size is SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis). Distinct peptide bands are obtained, enabling a qualitative measure of peptide profile. For hydrolysates, this technique can also be used to check whether there is still intact protein present. A fraction >10kDa determined in the MWD analysis may be caused by aggregates instead of intact proteins. Aggregates will be completely dissolved during SDS analysis, and no intact protein bands will be observed in the SDS-PAGE gel.
In the SDS-PAGE method, the solution of proteins to be analysed is first mixed with SDS, an anionic detergent which denatures (unfolds) secondary and non–disulfide–linked tertiary structures, and applies a negative charge to each protein in proportion to its mass4,5. Next to denaturation by SDS, also a reducing agent can be added, such as mercaptoethanol or DTT, which breaks down the disulfide bridges. Using SDS and reducing agent together will abolish all process induced covalent and noncovalent interactions of proteins. The samples are applied to an SDS-PAGE gel followed by applying of an electric current, causing the negatively charged proteins to migrate towards the anode. Short proteins will experience less resistance from the gel resulting in a higher migration speed. Larger proteins will migrate more slowly.
The proteins in the gel can be stained allowing visualization of the individual fractions. Different proteins will appear as distinct bands within the gel (see figure 3). Molecular weight markers can be used to estimate the masses of the protein fractions. SDS-PAGE is not suitable for small peptides.
figure 3 – SDS-PAGE stained with Coomassie Brilliant Blue. The lanes show the following samples from left to right: molecular marker, intact casein, a casein hydrolysate, intact whey and three different whey hydrolysates.
Free Amino Acids (FAA)
To measure the amount of free amino acids (FAA), the hydrolysate is derivatized with phenylisothiocyanate (PITC) and subsequently analysed by reversed phase high performance liquid chromatography (RP-HPLC)6. This separation, in conjunction with a standard profile will give the FAA amount for each amino acid individually. Typically, these results are expressed as the sum of the individual values: % total free amino acid content.
FrieslandCampina Domo developed a new technique called fingerprinting. Each hydrolysate has its own unique fingerprint. Even when two distinct hydrolysates have an equal MWD profile, their fingerprints will show their unique character.
The fingerprinting technique is based on a RP-HPLC technique, separating peptides based on their hydrophobicity and size. The more hydrophobic the peptides are, the later they elute from the column. In addition to this, when the peptide size becomes larger than ~1.5 Dalton, increasing size will also delay the elution. This fingerprinting technique is not only applicable for single hydrolysates, but also for hydrolysate characterization in complex matrices, such as infant formulas in which also carbohydrates and lipids and many other ingredients are present (figure 4). FrieslandCampina Domo uses this technique during new product development, for fine tuning and comparing specific properties of hydrolysates.
Figure 4- Detection of hydrolysate in pure form (light blue) and same hydrolysate in the complex matrix of an infant formula (dark blue).
United States Pharmacopoeia & the National Formulary: USP 23-NF18. 1995. Content of alpha amino nitrogen, p1338.
AOAC. Official methods of analysis, Washington DC:Horowitz, 1995, p7.
Adler-Nissen, J. (1986). Enzymic hydrolysis of food proteins. New York:Elsevier Applied Science Publishers. P9-17 and p. 146-147.
Shapiro AL, Viñuela E, Maizel JV Jr. Molecular weight estimation of polypeptide chains by electrophoresis in SDS-polyacrylamide gels. Biochem Biophys Res Commun. 1967;28(5):815-820.
Weber K, Osborn M. The reliability of molecular weight determinations by dodecyl sulfatepolyacrylamide gel electrophoresis. J Biol Chem. 1969;244 (16):4406-4412.
B.A. Bidlingmeyer BA, Cohen SA and Tarvin TL. Rapid analysis of amino acids using pre-column derivatization. Journal of Chromatography. 1984;336:93-104.