Pet Food Enzymes - Application Sheets

• Enzymatic Hydrolysis of Proteins Using Novozymes Proteases
• Extensive hydrolysis of proteins with Flavourzyme
• Use of Novozymes amylases in dry pet food extrusion
• Use of Proteases in Production of Digest for Pet Food

Enzymatic hydrolysis of proteins is a way to improve the properties of proteins. The properties of a protein hydrolysate are determined by the degree of hydrolysis (see below) and by the structure of the peptides produced. These in turn are dependent on the nature of the protein and the specificity of the enzyme used, as well as on the hydrolysis conditions, particularly pH and temperature.

Degradation of a protein renders it more soluble. Other functional properties, such as emulsifying ability, foaming capacity, viscosity, gelatinization and water absorption capacity are also affected by hydrolysis. The taste of a protein is affected by hydrolysis. This goes both for the flavour of the protein and for bitterness (please find more on taste development below). The nutritional value of a protein is normally maintained or increased by enzymatic hydrolysis, which is carried out under mild reaction conditions. The protein is simply broken down into smaller units: peptides and amino acids.

The following food-grade proteases are recommended for the hydrolysis of proteins:

• Endoproteases: Alcalase, Food Grade®; Neutrase®; Protamex®; Novo-Pro™ D
• Exopeptidase/endoprotease complex: Flavourzyme®

Endoproteases work by cleaving peptide bonds in the interior of polypeptide chains, whereas exopeptidases cleave off amino acids one at a time from the end of polypeptide chains. Figure 1 shows how the different proteases will hydrolyze different bonds in a protein.

When preparing protein hydrolysates, the typical protein contains 8-12%, and the typical enzyme dosage with endoproteases is 0.5-2% based on the weight of protein. The typical enzyme dosage with Flavourzyme is 10,000-25,000 LAPU/kg protein, corresponding to 20-50 kg of Flavourzyme 500 L or MG per ton of protein.

The ratio between enzyme and substrate determines the speed of hydrolysis, which is highest initially and decreases with time. The hydrolysis eventually comes to a halt when there are no more peptide bonds available for the enzyme. The maximum attainable degree of hydrolysis depends on the nature of the protein and the specificity of the enzyme.

The degree of hydrolysis (%DH), defined as the percentage of peptide bonds cleaved, is a key parameter which characterizes a protein hydrolysate:

%DH= (Number of peptide bonds cleaved/total number of peptide bonds) x100%

The %DH can be measured by various methods, e.g.:

• Increase in osmolality measured as freezing point depression
• Determination of free amino groups, e.g. by using the OPA method
• Titration in a pH-stat of the free amino groups generated
• Formol titration

Descriptions of these methods are available on request.

When insoluble protein is solubilized by proteolysis, this can be followed conveniently by measuring the dry matter content in the soluble phase, e.g. by measurement of °Brix.

The flavour (meaty, vegetable, etc.) generally increases in intensity as the protein is broken down, eventually into small peptides and amino acids. Degradation of proteins can result in the formation of bitter peptides.

Some bitterness will usually be present at intermediate degrees of hydrolysis. Bitterness is believed to be caused by the presence of peptides of a certain size with terminal hydrophobic amino acids, see for instance Figure 1.

After hydrolysis with endoprotease, short peptides are formed. Some of these have terminal hydrophobic amino acids and are thus bitter peptides. Further hydrolysis with exopeptidases breaks down these bitter peptides.

The tendency to bitterness therefore depends not only on the degree of hydrolysis but also on the structure of the peptides produced. For example, casein will, when hydrolyzed with Alcalase, start to get bitter even at a degree of hydrolysis of 1%. The tendency to bitterness is reduced substantially when hydrolyzing casein with Protamex, and further reduced when using Flavourzyme.

Optimal working conditions for the individual enzymes are summarized in Table 1, which also lists a number of characteristics of the protein hydrolysates produced by the enzymes.

The progress of hydrolysis with different Novozymes proteases is illustrated in Figure 2.

The choice of enzyme for a given application depends on the substrate and the desired properties of the final hydrolysate. More detailed Application Sheets are available for several specific applications, and we would be pleased to help you identify the optimal enzyme(s) for your particular application.

In order to control the functional properties of the hydrolysate it may be important to stop the enzyme reaction at a closely defined %DH value. All the proteases can be irreversibly inactivated by heat treatment. Table 2 suggests treatment times for inactivation at a given pH and temperature. However, inactivation by heat treatment is very much dependent on the substrate (substrate concentration, pH, etc.). Thus, the documentation for efficient elimination of protease must be based on actual analysis for the detection of residual activity.

The proteases Alcalase, Neutrase and Protamex are all inactive at pH 4 or below. The reaction can, therefore, be stopped instantaneously by the addition of a convenient acid, e.g. hydrochloric, phosphoric, malic, lactic or acetic acid.