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

Flavourzyme can be used to degrade a variety of food proteins extensively, i.e. to degrees of hydrolysis of up to 70% (degree of hydrolysis = percentage of peptide bonds broken).

Extensive hydrolysis of proteins with Flavourzyme creates savoury protein hydrolysates, without the bitterness that often occurs with moderate enzymatic hydrolysis. Also, the potentially harmful substances formed by acid hydrolysis are avoided and the protein hydrolysate is low in salt.

The protein should be in a physical form which makes it accessible for enzymatic degradation. Many substrates will already be in such a form, e.g. flours, concentrates and isolates of vegetable proteins, casein, whey, etc. Other substrates, such as meat, bone and soya flakes, will require suitable pretreatment.

Heat treatment (e.g. pasteurization or pressure-cooking) of the protein prior to hydrolysis will denature the protein, making it more accessible, and will also help to prevent microbiological spoilage during hydrolysis, which may last many hours.

Flavourzyme contains a number of different proteolytic activities working in the neutral to slightly acidic pH range. Optimum utilization of Flavourzyme can therefore be achieved by hydrolyzing at various pH values in this range as described below. In general, two different hydrolysis procedures are recommended, depending on the initial pH of the substrate mixture to be hydrolyzed:

In this case, initial hydrolysis with Protamex™ or Alcalase® is recommended before hydrolysis with Flavourzyme:

1. First hydrolysis with Protamex or Alcalase starting at the pH of the substrate mixture.

Substrate conc........................8-16% protein

Temperature..........................55°C (131°F)

Enzyme dosage......................6-15 kg Protamex 1.5 MG or

Enzyme dosage......................4-10 kg Alcalase 2.4 L per ton of protein

Time.......................................15-30 minutes

The pH should be allowed to drop by itself during hydrolysis, typically by one pH unit.

2. Second hydrolysis with Flavourzyme. The pH will typically drop further to a pH of around 5.5.

Temperature............................55°C (131°F)

Enzyme dosage........................10-25 kg Flavourzyme 1000 MG

Enzyme dosage........................per ton of protein

Time........................................4-16 hours

It should be noted that hydrolysis with Alcalase in the first step will in some cases yield a relatively bitter protein hydrolysate due to the formation of bitter peptides. This bitterness will disappear during the subsequent hydrolysis with Flavourzyme, as the bitter peptides are degraded. There may also be some clotting of the protein during the first hydrolysis with Protamex or Alcalase, but this will also disappear as the hydrolysis of the protein progresses further.

In this case, a one-step hydrolysis with Flavourzyme is recommended:

1. First hydrolysis with Flavourzyme starting at the pH of the substrate mixture. The pH will remain fairly constant during hydrolysis, as most amino acids have PKa values in the slightly acidic range.

Substrate conc...........................8-16% protein

Temperature..............................55°C (131°F)

Enzyme dosage.........................10-25 kg Flavourzyme 1000 MG

Enzyme dosage.........................per ton of protein

Time..........................................4-16 hours

A higher degree of hydrolysis can in some cases be obtained by increasing the pH to 7.0 after a few hours of hydrolysis and adding an additional 10-25 kg Flavourzyme 1000 MG.

Suspending the protein, especially at high substrate concentrations, may often be difficult due to high viscosity. In this case, suspension can be facilitated by first preparing a solution of the enzyme to which the protein is then added while stirring gently. The protein should be added immediately after the enzyme solution has been prepared, so that the enzyme does not degrade itself.

The long reaction times, which are favourable for the formation of flavour, will in some cases require that the reaction mixture is preserved or stabilized to prevent microbial spoilage. Flavourzyme tolerates salt relatively well. The activity is only diminished by around 10% in the presence of 5% NaCl.

The reaction mixture should be stirred adequately to facilitate hydrolysis. Suction of air into the reaction mixture should be avoided during stirring, as this could lead to excessive formation of foam.

The progress of hydrolysis can be followed, e.g. by measurement of osmolarity.

Fresh meat and bone (or bone cake from mechanical recovery of meat) can be used to produce brothy protein hydrolysates. Meat should be minced prior to hydrolysis in order to make it accessible for enzymatic degradation, and bones should be chopped into pieces a few centimetres in size for the same reason.

The pH of the substrate mixture will typically be around neutral and so procedure A above should be used. When bone (or bone cake) is used as the substrate, the ratio between bone and water added should be approximately 1:2.

Casein, whey and skim milk can easily be hydrolyzed with Flavourzyme. The initial pH will normally be around neutral and so procedure A above should be used.

Skim milk will yield a cheesy flavour, whereas casein and whey hydrolysates will have a milder taste, suitable for dietary products amongst others. No bitterness will normally be present, not even in caseinate hydrolysates, if hydrolyzed extensively with Flavourzyme.

Commonly used vegetable proteins such as soya, maize and wheat gluten can also be hydrolyzed using Flavourzyme, yielding savoury hydrolysates.

Vegetable protein may often be associated with carbohydrate, e.g. plant cell wall material. Flavourzyme contains amylase and other carbohydrase activities and will hydrolyze starch and certain other carbohydrates to some extent.

However, in some cases pretreatment of the vegetable protein source with Viscozyme® may be needed to release the protein, especially when less refined raw materials are used. For further details, please see the Viscozyme Product Sheet.

Soya will typically have a pH around neutral when suspended in water, in which case procedure A should be used. Maize and wheat gluten will typically have a slightly acidic pH, in which case procedure B should be used.

Most other proteins, such as gelatine, fish, peas, yeast, etc., can also be degraded by Flavourzyme, yielding hydrolysates rich in taste and free from bitterness.

Substrate conc.:  8% protein

Enzyme conc.:  37 LAPU/g protein

Initial pH:  7.0 (no adjustment of pH during hydrolysis)

Temperature:  50°C (131°F)

Method:  TNBS

The enzymes can be inactivated by heating at 85°C (185°F) for 5 minutes or at 120°C (248°F) for 5 seconds after the hydrolysis has been completed.

Whereas traditional acid hydrolysis, which is carried out at extreme pH and temperatures, creates a number of flavoured by-products, the mild reaction conditions during enzymatic hydrolysis prevent the formation of such byproducts. The flavour of the protein hydrolysate can be developed further by heat treatment, e.g. with reducing sugars to form Maillard products.

Various separation steps may be needed in order to remove unwanted materials. Preservation and further formulation may also be carried out, depending on the requirements for the final use of the protein hydrolysate.