Introduction of Protein Modification
Protein modification is found in all cells and tissues of all living things, including plants. All organisms need protein. Plants are the source of the protein in the human diet. Protein plays many roles in food. Protein is essential for nutrition. Protein provides structure and texture to foods. Protein influences the way food interacts with water and fats. Protein can thicken and firm foods.
Plant and animal proteins have many of the same roles, but plant proteins do not always perform optimally to fulfill these roles. Improving the solubility and stability of proteins improves the ability of proteins to perform their roles.
Modification of food proteins improves the ability of these proteins to perform their roles.
Improvement of proteins to perform a role in foods requires amino acid sequencing of the protein to better determine how the protein will interact with other food ingredients. Modification of proteins to fulfill a role increases the quality of food and the outcome desired.
The purpose of this paper is to define protein modification and provide examples of the methods of protein modification. Other topics discussed in this paper are the limits of protein quality and functionality, the problems associated with the use of proteins in foods, and the solutions to overcoming these problems.
What are some effects of protein structure?
- Solubility
- Oil and Water Absorption
- Emulsification and Foaming
- Gelation
- Heat Stability
- Digestibility
- Other Effects on Food
- Nutritional Availability
- Protein Structure and Function
What determines Protein Quality?
The quality of a protein is broad and includes factors such as the following.
- Amino Acid Composition
- Purity
- Functional Properties
- Digestibility
- Heat and Oxidative Stability
- Solubility
- Other Quality Attributes
- Color and Flavor
- Processing Stability
- Microbiological Quality
For food and beverage applications, a protein’s functional properties are as important as its amino acid composition for nutritional quality.
The quality of a protein is broad. Therefore, the statement “protein X is of better quality than protein Y” must be evaluated for the specific application.
Factors Affecting Protein Structure
- Primary Structure
- The order of amino acids within a protein.
- Secondary Structure
- Arrangements of amino acids within a protein
- Tertiary Structure
- The three-dimensional configuration of an individual protein
- Quaternary Structure
- The arrangement of multiple protein subunits.
Protein Modifications
There are multiple levels of protein structure. Alterations of a protein can occur at any of these levels. Changes to protein structure and function can occur from exposure to heat and acid or base. Other alterations can occur from exposure to enzymes. The goal is to intentionally modify protein structure and function while avoiding unwanted side effects.
Physical Protein Modification
Heat Treatment
Heat treatment of proteins can be achieved by exposing them to heat to break down the intermolecular bonds that stabilize their structure. This process can result in partial or total unfolding of proteins.
Partial unfolding of proteins can be of beneficial effect on the functional properties of the proteins. For example, new interfacial activities of previously buried hydrophobic groups may enhance foam and emulsion stability.
Thermal treatment can improve the digestibility and safety of foods. However, the treatment may cause the formation of protein aggregates.
Thermal treatment should be optimized so as to achieve partial unfolding of the proteins instead of their complete denaturation.
High Pressure Treatment
Structural changes that occur to proteins under the influence of high pressure may include protein unfolding, aggregation and gelation.
Compared to other treatment methods, high pressure treatment of food products may help maintain the structure and other physical properties of heat sensitive foods.
The final effect of high pressure treatment depends on the pressure, time, and temperature of treatment as well as the protein concentration.
Ultrasonication
Different kinds of drying processes (e.g., spray drying, freeze drying) can be used to modify the structure and function of proteins. Different conditions can affect the solubility and stability of the protein.
A processing strategy should be developed to minimize the undesirable effects of the drying process. The objective should be to develop a stable dried product of the protein.
Thermal Protein Modification
Proteins can be modified by thermally induced changes in their structure. Proteins are made up of long, flexible chain-like structures which make up the final three-dimensional structure of the protein. One of the functions of these long, flexible structures is to hide groups within the protein. Heating a protein can cause the long, flexible structures to lose their shape and thus unfold.
This process makes previously hidden groups available to participate in reactions. Heating can also cause other changes in protein structure which can lead to improved functional properties of the protein. Thermal treatments are widely used in food processing. Texture, moisture retention and gel forming ability of food proteins can be improved by thermal treatment. Like other extreme treatments, excessive heating of food can cause loss of nutrients, and undesirable change of color and flavor.
Enzymatic Protein Modification
Enzymatic protein modification employs proteases to effect specific changes on protein molecules and potentially other substrates, to yield smaller peptides or products of other defined structures. Depending on the goal of the modification, improved functionality of the protein can be achieved. Like chemical modification, the effects of the enzymatic modification are generally irreversible. However, the process is generally more controlled.
For example, the characteristics of the enzymes, pH, temperature, time, protein concentration, and the extent of the hydrolysis, among others, can be altered to achieve the desired effect. Complete hydrolysis of the protein can however result in small peptides that may be bitter, and may also effect other undesirable changes (e.g. reduced gel strength, foam stability, etc.). It is thus important to control the extent of the hydrolysis, and to achieve an appropriate balance to ensure that the modified protein retains the required functionality and does not gain any new undesirable properties
Chemical Protein Modification
Chemical Protein modification is the alteration of the properties of a protein via chemical means. One common example is phosphorylation. The charge on a protein can also be altered by a process called deamidation. A protein’s hydrophobicity can be changed by acylation or succinylation. Protein cross-linking is a process that is becoming increasingly common in food protein modification. Gelation of food protein can also be achieved via protein modification. Functional groups of proteins can be changed by chemical protein modification. These modified groups can be specifically altered to satisfy the desired functionality for a given food system.
Some examples of desirable changes to food protein systems by chemical protein modification are solubility and emulsifiability. Undesirable side effects to chemical protein modification are loss of functionality of the protein as well as undesirable changes to the overall protein. It is imperative that the processing chemicals used in food comply with food processing laws. Adequate control of numerous processing variables (reagents, pH, temperature, time, moisture, and extent of modification) is essential to ensure desired protein modifications.
Protein Modification by Microbial Fermentation
Microbial fermentation is a bioprocessing technology that is naturally modifying food system components by using specific food-grade microorganisms and their enzymes. Proteolytic enzymes produced by microorganisms can break proteins to smaller peptides. Several microbes can modify the structure of proteins by modulating the pH of the medium and producing different types of metabolites. The utilization of microbial fermentation of plant proteins including soy, pea, and chickpea, may result in improved protein functionality, and may reduce unacceptable flavors and odors.
The criteria that control the success of the fermentation process include the type of microorganism, time, pH, moisture, and other components of the substrate. Undesirable flavor, inconsistent texture, and other unacceptable qualities may occur due to poor control of the fermentation process. Maintaining hygiene and controlling the process conditions may improve the consistency and the overall quality of protein modified by microbial fermentation
Protein Hydrolysis and Improvement in Quality
For improvement in quality, one of the methods that may be employed is hydrolysis.
Hydrolysis may be accomplished using enzymes, acids or bases.
Of the above methods, use of enzymes guarantees greater process control.
Effect of controlled hydrolysis
This may improve:
- The protein’s ability to disperse in a medium
- Protein solubility
- The water-holding capacity of protein
- Ease of protein digestion
- May affect the protein’s physical properties for processing
- Effects of uncontrolled or excessive hydrolysis
This may result in undesirable sensory qualities and gel weakness.
It is important to totally control the extent of protein hydrolysis in order to achieve the desired improvement in the functionality of the protein.
Effect of controlling the pH of proteins
At the isoelectric point of a protein, the protein is not soluble because of weak repulsion between protein molecules.
Past the isoelectric point, the protein is negatively charged and more soluble.
To achieve functional improvements, pH of protein solutions may be adjusted to a value within the range of the desired functional improvement.
Inappropriate control of pH may result in denaturation and irreversible changes in the protein.
Optimization of Wheat Proteins
Plant proteins, including wheat proteins, can be used in food products. Many aspects of plant proteins, including solubility, emission of beany flavors, and poor gelation, could limit their use. Several approaches can improve the functional properties of plant proteins. This includes physical or chemical modification of plant proteins.
Some of the physical and chemical methods to modify plant proteins include the use of heat, high pressure, chemical and enzymatic treatments, pH modification, fermentation, ultrasound, and protein-polysaccharide interactions. The most appropriate modification method depends on the type of plant protein. The method must also be optimized to avoid adverse effects.
Potential functional ingredients can be developed using different methods to covalently link proteins to other food components. These methods can generate ingredients with unique functional properties. However, these ingredients must be tested for potentially adverse impacts on the safety and/or nutrition of foods.
How to Manage Protein Modification
Process Control
Process control is crucial in the context of protein modification to attain dependability and efficiency in implementing changes to the protein of interest. Modifying proteins can be carried out using a variety of physical, chemical, enzymatic and/or biological methods. Among all the variables that need to be tightly controlled during a protein modification process, some of the key variables may include the processing temperature, pH, time, relative humidity, and concentration and activity of the modifying agent(s). Protein modification processes should be carefully optimized to reduce unwanted by-products and ensure reproducibility. Identifying and resolving processing issues at an early stage allows a protein modification service provider to deliver high quality proteins with the desired functionalities and activities to a client.
Each modification must be aimed at a specific goal.
Each modification of a protein should be strategically planned. Things to consider may include improving protein solubility, water holding capacity, or other functionalities. It may be used to improve the stability of a protein during processing or storage by altering the protection of the protein from the environment. Sometimes, the same modification may be used to enhance the functional property of the protein and its digestibility. Prior to modifying a protein, it is important to analyze the protein to assess its purity, identity, molecular and functional properties, and its potential impact on human health.
It is important to understand the limitations of the starting material to identify the best modification strategy to protect the protein from unfavorable changes that may adversely impact its usefulness. Processing conditions may also be determined based upon the comparison of the modified protein to the unmodified control. The comparison to the control may allow researchers to determine what conditions of the process may favor the desired modification and help them avoid protein modification in cases when the modification may adversely impact the quality of the protein.
Identify the unmodified protein
Processing of protein ingredients results in changes in the physical and functional attributes of the ingredients. The extent of changes caused by protein modification can only be assessed in relation to the unmodified protein. Therefore, prior to any protein modification, thorough analysis of the unmodified protein is essential.
Determine Moisture of protein source
The level of moisture present in a protein ingredient can affect the outcome of protein modification. Moisture can affect the concentration of components and also impact the temperature at which the protein ingredient is processed. A high level of moisture in a protein ingredient can provide an environment for undesirable chemical and microbiological reactions. Removing moisture from a protein ingredient can affect the solubility and functionality of the protein. Thus, it is essential to adequately control the level of moisture in a protein ingredient to ensure the desired outcome of a protein modification process.
Determine Fat in protein Source
Fat can alter the behaviors of proteins during processing. The presence of fats can also change the flavor of processed foods. Incorporation of fat can also increase the potential for food spoilage. It is important to remove fat from a protein source prior to it being used to manufacture foods. Lipids tend to bind with proteins and alter properties such as solubility and emulsification. For these reasons, it is important for food processors to measure the fat content of a protein source. It is particularly important that processors of protein ingredient products understand the fat content of the proteins they utilize so that they can produce a consistently excellent product.
Determine Ash in Protein Source
Ash, the inorganic residue, provides information about the mineral content and the overall level of impurity in a protein source. The presence of higher levels of ash may reflect poorer quality of the protein source. Ash content can also aid in identifying proteins which contain dissociated ions. Measuring ash content of a protein source prior to any chemical or physical treatments provides an analysis of the protein source prior to alteration and can aid in defining changes that may have occurred during the treatment.
Identify Carbohydrate Content in Protein Sources
It is important to analyze the carbohydrate content of a protein source as carbohydrates, like protein, can affect the physicochemical properties of a protein-based ingredient. Different types and levels of carbohydrates can affect the viscosity and the degree of water binding of a protein. Carbohydrates can also impact the stability of a protein and its tendency to coagulate. Carbohydrates may also bind to proteins and affect the functionality of a protein-based ingredient. Understanding the carbohydrate content of a protein allows the analyst to foresee the effects of processing and modification of the protein and to establish the limit of safety for processing and modification of the protein.
Functional properties
- What functional improvements are desired?
- Better solubility?
- Better emulsions?
- Better gels?
- Better digestibility?
- Heater stability?
- Texture?
- Other desires?
- What modification method will result in the desired functional improvements?
Protein Modification: Limitations and Considerations
There are several considerations when making modifications to protein.
- Reaction temperature can impact the degree of modification and denaturation.
- High pH can increase the rate of reaction, decrease protein solubility, and inactivate enzymes.
- Low moisture can decrease the rate of reaction, increase protein density, and increase aggregation.
- High protein concentration can increase the rate of modification and gelation.
- High enzyme concentration can increase the rate of hydrolysis.
- Processing must be of sufficient intensity to achieve the desired change.
Problems Associated with Protein Modification
There can be a number of reasons for failure of protein modification.
Temperature Control Issues
There can be over-heating and under-heating issues during the modification.
Overheating can lead to irreversible aggregation, loss of solubility and/or production of bitter peptides.
Under heating can lead to loss of activity due to improper folding.
pH Issues
There can be issues during adjustment of pH of the system.
Alkaline pH can lead to denaturation of the protein, loss of activity and precipitation.
Acidic pH can lead to partial hydrolysis of the protein and/or production of undesired by-products.
Chemical Treatment Issues
There can be issues due to excessive chemical treatment.
Treatment can lead to loss of nutritional value, loss of activity and/or change in physicochemical properties.
Other Issues
There can be issues due to lack of uniformity in drying and other post-modification treatment of the proteins.