Introduction on Protease
Protease is an enzyme that causes the chemical breakdown of proteins into smaller fragments, i.e. peptides or amino acids. In baking, especially with the use of wheat flour, protease breaks down the gluten present in the flour which helps in improving the working properties of the dough, i.e. prolongs the period of fermentation and proofing, alters the texture of the crumb, improves the volume of the loaf, and alters the overall quality of the baked product.
There are a wide variety of baked products that utilize protease e.g. breads, rolls, crackers, biscuits and cookies. However, there are a number of factors that affect the performance of protease e.g. type of flour, condition of the dough i.e. hydration, pH, and temperature of the dough, time and method of mixing, time and method of proofing, type of protease used, etc. If too much or excessive protease is used, it may break down the gluten too much to the extent that the dough loses its ability to retain the gasses of rising, resulting in a lower volume loaf with an open and fluffy crumb.
What is Protease?
Proteases or proteinases, are a group of enzymes that help breakdown proteins by cleaving the peptide bonds. The overall reaction involves the breakdown of larger proteins into smaller peptides or amino acids.
Gliadin and glutenin are two of the gluten proteins found in wheat flour that help contribute to the viscoelastic nature of gluten. The breakdown of these proteins helps breakdown the system that provides gas retention and dough strength. Protease helps alters this system by partially breaking down protein chains.
This can help the dough:
- More easily be shaped or rolled out
- Be less stiff
- Have less breakdown resistance
- Be more easily machinable
- Less shrink during sheeting
- Be more acceptable for some biscuit and cracker production
If too much is added, the gluten network will become weak and break down.
Protease in Bakery Products
The function of adding protease to a particular bakery product is determined by what kind of product is being made.
Bread
If too much gluten is developed in bread, the loaf may have a loose and open crumb. Protease is added to bread to adjust the rheology of the dough.
Other Products
If the product is not expected to have a lot of gluten development, like biscuits and cookies, the addition of gluten protease would enhance the sheeting of the dough, and improve shape and size retention, and the texture of the baked product.
Wheat Gluten
The use of protease is based on the characteristics of wheat gluten.
Wheat flour contains two major classes of gluten proteins.
- Gliadins
- Glutenins
Of the two, glutenins are more sensitive to mixing and contribute more to the elasticity of gluten and the strength of the dough.
The interaction of gluten proteins is brought about by the hydration of the proteins when water is added to the flour and mechanical mixing.
CO2 produced by fermentation becomes trapped in the gluten network and helps raise and expand the dough.
Balance of Gluten Strength
- Too much gluten strength causes the dough to be very tough and hard to work.
- Too little gluten strength causes the dough to lose its ability to trap gas.
- The goal of using protease is to adjust gluten strength to improve workability of the dough.
- The Effect of Protease on Dough
- There are many effects of using protease in dough.
Effects on Extensibility of Dough
- The most important of these effects is the ability of the dough to stretch.
- Protease treatment allows the dough to be stretched further without tearing.
- It allows the dough to be rolled more easily.
- Treatment with protease can also allow the dough to relax and more easily assume the desired shape.
- Protease can also weaken dough enough to improve workability.
- Not all effects of using protease are positive. Using too much protease to weaken the dough can reduce the ability of the dough to trap the gas being produced during fermentation.
Dough Relaxation can Affect:
- Sheeting
- Molding
- Cutting
- Forming
- Shrinkage
Determines the Effectiveness of a Protease
Several factors affect the performance of a protease. These factors are discussed below.
Quantity of Enzyme
Dosage is one of the most crucial factors.
Generally, more protease results in more activity. However, from a practical viewpoint of baking, this relationship may not always be the case.
Increasing the quantity of protease added to dough may not always be beneficial, and may, in fact, result in over-relaxation of the dough.
Therefore, to determine the optimal quantity of protease to add to dough, a dose response study is warranted.
According to a trial conducted by a baking concern, the following levels of a commercial protease were assessed:
- -Control
- -Low
- -Medium
- -High
- -Very High
From a regulatory and manufacturer’s activity specification point of view, the actual quantities of the enzyme added to dough in a baking trial should be specified. A universal value of a gram of enzyme per kilogram of dough cannot always be justified.
pH
All proteases are influenced by pH. Each has a range of activity. If the pH is within the range of the activity of the protease, it will be active and protein will be broken down. If the pH is not within the range, the protease will not be active and protein breakdown will not occur.
Dough ingredients can affect the pH of the dough. Among these ingredients are:
The type of flour used
The type of flour used:
- Yeast
- Salt
- Acids
- Milk Ingredients
- Chemical leavening agents
- Fermentation products
- The performance of a protease can change with any adjustment of these ingredients.
Among the different proteases, some have a broad range of activity while others are more sensitive. It is always a good idea to test the performance of the protease in the final product.
Temperature
How fast an enzyme-catalyzed reaction happens depends on how warm or cold it is.
As long as the temperature of the reaction isn’t too high, the active site of the enzyme is not changed, and the enzyme is able to still function.
At extremely high temperatures, all proteins, including enzymes, can be denatured.
Thus, in the case of the example of the use of the enzyme, protease, during different stages of breadmaking (i.e., mixing, resting, fermenting, proofing, baking, etc.), the major effect of the protease during the early, heat-sensitive steps is of greatest importance.
During the early stages of breadmaking, if the enzyme acts quickly during mixing and proofing, the amount of change the enzyme brings about is greatly enhanced.
On the other hand, if the enzyme acts too quickly during earlier steps of breadmaking, the changes brought about by the enzyme will likely be damaging to the final product.
Mixing Time
During the mechanical mixing of bread dough that contains protease, gluten development and the breakdown of gluten by the enzyme occur at the same time.
Therefore, the final properties of the bread dough depend on the interplay of the mechanical work and the breakdown of gluten by the enzyme.
Lengthening the time of the mechanical work can strengthen the bread dough, but too much work can break down the dough and make it too weak to support the shape of the final product. In the presence of a protease, the optimal time for mechanical work is less than for a dough that does not contain the enzyme
Dough Hydration
Water is a necessary ingredient for the action of a protease because it provides the environment for the action of the enzyme.
Factors affected by water content of the dough include the mobility of the proteins, the development of gluten, and the overall consistency of the dough.
Because of the effects of water content on proteins and gluten, the effects of a protease added at one water level cannot be automatically assumed for a different water level.
Salt
Salt impacts the functioning of gluten and the rheology of dough.
The inclusion of salt in a formula strengthens dough.
Salt helps to manage the effects of proteolysis.
It is worth noting, a more concentrated salt solution will result in a stronger dough.
The reverse is also true.
The relationship and effect achieved by varying the levels of salt and active protease should be considered when formulating.
It is important to consider the dough’s commercial context when making final formulation decisions.
Fermentation Time
Fermentation time is closely tied to the activity of a protease.
A longer fermentation time will result in greater modification of the proteins in a dough.
If a protease is active for an extended period, it is possible for a dough to lose the ability to retain gas, resulting in spreading and loss of machine-ability.
It may also result in collapse during proofing.
Dough Rising Time
There are a number of commercial dough products where a resting period is required.
During resting time, dough relaxation occurs and proteolysis may occur.
It is possible to observe the effects of proteolysis as a result of the relaxing of dough.
If the effect of resting on dough has not been previously evaluated, it may be worth assessing the effect dough has at the end of its resting period.
Enhancing the Function of Proteases in Bread
A high level of activity of a protease results in uncontrolled and excessive modification of bread.
Generally, bread formulators aim to achieve a controlled level of modification.
An effective strategy is as follows:
Characterize the Flour
Protein Content
Flour type is based on the protein content of the wheat from which it is milled. Proteins combine with water to form the gluten network during kneading and improve dough’s viscosity. A higher protein content leads to stronger, more elastic dough that is able to retain gas and results in a higher rising of the final product. Protein content is inversely related to protein function. Therefore, to achieve a tender product, a lower protein flour is used.
Gluten Strength
The strength of the gluten network has an effect on the structure and volume of the final product. For bread, the gluten network needs to be strong to withstand mixing, fermentation, and rising. A strong gluten network also allows the dough to maintain and capture more carbon dioxide. The strength of the gluten network is related to the protein content.
Falling Number
Falling number tests measure the level of active amylase in the flour and how quickly starch gelatinizes during baking. The higher the Falling number, the less the amylase activity and the slower starch gelatinization, which leads to a longer rising time. A lower Falling number means starch gelatinizes quickly and the protein network breaks down.
Water Absorption
Falling number tests measure the level of active amylase in the flour and how quickly starch gelatinizes during baking. The higher the Falling number, the less the amylase activity and the slower starch gelatinization, which leads to a longer rising time. A lower Falling number means starch gelatinizes quickly and the protein network breaks down.
Dough Rheology
The physical properties and behavior of a dough during the various stages of its formation from mixing to stretching and from fermenting to final bake is referred to as the rheology of the dough. The rheology of a dough provides valuable information on the behavior of the flour being processed and how the dough will behave during processing and impact the final product.
Establish a Control
- Mixing Time
- Dough Temperature
- Dough Consistency
- Proof Time
- Volume of Risen Bread
- Structure of Bread Crumb
- Texture of Bread Crumb
Perform a Dosage Trial
Examine the effects of various levels of the protease used in this trial.
Evaluate Dough
Assess the following for each of the trials:
- Resistance to Stretch
- Stickiness
- Relaxation
- Gas Bubbles
Evaluate Baked Bread
Determine the following for each of the breads prepared:
Specific Volume of the Loaf
The specific volume of a loaf is the volume of the loaf in relation to its weight. A high specific volume signifies the dough has undergone adequate fermentation, has a good gluten structure and is able to retain gas during the process. A loaf with a low specific volume may be the result of a number of factors including, under fermentation, a weak gluten structure, or a poor gas holding capacity.
Fitness of Bread Crumb
Firmness is the amount of force needed to compress the bread crumb. The softer the bread, the lower the firmness. Firmness can be determined by a texture analyzer or by a judge through a sensory test. The crumb of bread fresh from the oven is fairly soft. With the loss of moisture, the crumb becomes firmer. If the starch of the flour has fully gelatinized and then set, or if the gluten has developed too much, the crumb will become dense. The crumb should become firm enough to support the slice of bread, but not too firm. The softer the crumb the better.
- Evenness of Bread Crumb
- Moisture Content
- Bread Texture
The ideal amount of protease would be that which allows the required changes in the behavior of the dough and still allows the structure of the dough to be maintained.
Resistance and Extensibility of Dough
Texture Analysis
Firmness, Springiness, Chewiness and Cohesiveness can be measured objectively. Sensory evaluation of baked goods can be subjective. Therefore, in situations where a clear distinction cannot be made in sensory evaluation, an objective measurement can be employed.
Sensory Evaluation
In the case of bread, assessment of aroma and slice appearance can be made in conjunction with instrumental measurement. Softness and chewiness of bread can also be determined through sensory evaluation.
Mouthfeel can be measured objectively and can be used to determine the quality of biscuits. Tenderness, crispness and other attributes can be assessed through mouthfeel. Flavor and aftertaste of biscuits can also be evaluated through sensory means.
A treatment which improves the workability of a dough can give an undesirable sensory effect and, therefore, should not be considered an effective formulation.
Modification of Dough through the action Protease and Yeast
There are several effects which can be attributed to the over-relaxation of gluten. One of which is stickiness of the dough which can occur through excessive action of the enzyme.
There are other factors which can lead to stickiness of dough. Some of these include the use of excessive water in the dough, elevated temperature of the dough, use of high- extracted flour, damaged starch in flour, excess fermentation, presence of certain enzymes, and/or other additives. Therefore, an evaluation of other factors should be made before concluding that stickiness is attributable to the action of a protease.
Common Problems and Solutions
Problem 1: Not enough effect from the added protease
Possible reasons:
- Inadequate dosage
- Limited enzymatic activity
- Poor distribution of enzyme in the dough
- Insufficient time for the reaction to occur
- Incorrect pH of the dough
- Dough is too cool
Possible solutions
Check enzymatic activity and adjust exposure time or dosage (within the limits given by the enzyme supplier).
Problem 2: Dough is too soft
Possible reasons:
- Too much enzyme added
- Dough rested too long
- High dough temperature
- Using a high-protein flour
- Protease is too strong
Possible solution
Reduce the activity of the protease.
Problem 3: Dough is too sticky
Possible reasons:
- Too much protease added
- Dough temperature is too high
- Too much water added to the dough
Possible solution:
Evaluate enzyme dosage and determine if this was the cause of sticky dough.
Problem 4: Volume of the loaf is too small
Possible reason:
Gluten has been weakened too much
Possible solution:
Decrease the activity of the protease and monitor the strength of the dough.
Problem 5: Biscuit dough is too strong
Possible reason:
Limited activity of the added protease
Possible solution:
Improve the activity of the protease and monitor the strength of the dough.