Introduction of Amylolytic Enzyme
Amylolytic enzymes play a significant role in food processing. The term ‘amylolytic’ is derived from the breaking of starch, known as amylolysis. Amylolytic enzymes break starch and other linked carbohydrates into smaller units including dextrins, maltose, maltotriose and glucose, as well as other oligosaccharides.
Starch is one of the most abundant carbohydrates in food. Starch is found in numerous foodstuffs including wheat, rice, corn, barley and other cereals, as well as root crops such as potatoes and cassava. Even though starch is a good energy source, at times it poses problems in food processing due to its nature and properties. Amylolytic enzymes are an invaluable aid in controlling starch related problems as they help to improve the quality, texture, taste, and overall digestibility of food, and help to ease processing of many foodstuffs.
The main amylolytic enzymes are α-amylase, β-amylase, pullulanase, glucoamylase and others. The action of these enzymes on starch varies and, through proper selection and adjustment of controlled conditions, food processors are able to achieve the desired degree of starch hydrolysis.
What Is Starch?
To understand amylolytic enzymes, some background on starch is helpful.
Starch is a polysaccharide made of glucose. Starch is of two major types: amylose and amylopectin. Each of these is described below.
Amylose
Amylose is mainly a linear chain of glucose joined by α-1-4 glycosidic bonds. Being a relatively linear structure, amylose is of great importance in the firm structure of starch.
Amylopectin
Amylopectin is highly branched. This is the structure that it contains:
Main chains: α-1,4 bonds
Branching points: α-1,6 bonds
The branched structure of amylopectin makes complete starch hydrolysis more complex since many enzymes cannot efficiently break α-1,6 bonds.
This is the reason why many food processors use a combination of different amylolytic enzymes.
What Are Amylolytic Enzymes?
Amylolytic enzymes are enzymes that break starch down into smaller carbohydrate molecules.
It occurs through hydrolysis, thus, the breaking of glycosidic bonds with the help of water.
A simplified reaction can be:
Starch + water → dextrins + oligosaccharides + maltose/glucose
The products of a reaction depend on the enzyme used and the control of the reaction.
Amylolytic enzymes are biological catalysts that speed up the reaction but are not consumed during the reaction like a normal substrate would be.
Major Types of Amylolytic Enzymes
α-Amylase
α-Amylase is one of the most important amylolytic enzymes in food processing.
It is an endo-enzyme meaning it acts on the internal α-1,4 bonds of starch chains.
For example:
Long starch chain → shorter chains → dextrins
Since it acts on the internal bonds, one of the first effects is on starch solution viscosity. It lowers the viscosity which is known as liquefaction.
Importance in food processing
- α-amylase has many uses in:
- Baking
- Brewing
- Production of maltodextrins
- Fermented foods
- Various animal-feed industries
α-amylase, in conjunction with starch in bread, helps to produce fermentable sugars.
In conjunction with starch, α-amylase can help to produce sugars in bread that are fermentable by yeast.
β-Amylase
β-Amylase breaks down starch chains in a stepwise manner as opposed to the random manner in which α-amylase operates.
This makes β-Amylase an exo-acting enzyme.
It breaks down starch chains by removing successively maltose units from the non-reducing ends of starch chains as opposed to α-amylase which randomly breaks the starch chains to release individual maltose units.
β-Amylase’s function is to produce maltose.
The general reaction can be simplified in the following way:
Starch………………………………………… →…………… Maltose……………..+……………. Shorter Chains
β-Amylase is important in the following industries:
- Brewing
- Production of maltose
- Malting
- Some cereal processing.
β-Amylase cannot digest highly branched amylopectin, therefore, the complete conversion of highly branched amylopectin requires the action of other enzymes.
Glucoamylase
Glucoamylase, also known as amyl glucosidase, breaks down starch and related dextrin’s and releases glucose.
Glucoamylase attacks starch chains from their ends and slowly breaks down α-1,6 bonds.
Generally, it breaks down α-1,4 bonds.
Glucoamylase is very important in industry for:
- production of high glucose syrups
- starch hydrolysis
- production of fermentable sugar
Glucoamylase’s action is very important in the following industries:
- Fermentation
- Brewing
- starch hydrolysis
Pullulanase
Pullulanase breaks the α-1,6 bonds, which leads to an unbranched amylopectin chain.
This function makes pullulanase very important when high degrees of starch hydrolysis are required.
It simplifies the extension of the amylopectin chain so that other enzymes can easily hydrolyze the chains.
Pullulanase is used with additional α-amylase or glucoamylase as other enzymes to complete starch conversion.
How Do Amylolytic Enzymes Work?
Like other enzymes, the binding site for amylolytic enzymes substrates are active sites.
The primary substrate for amylolytic enzymes is starch or a product derived from starch.
The main process can be outlined as:
Step 1: Contact of enzyme and starch
The enzyme and starch molecules come into contact.
Step 2: Binding
The starch chain binds to the enzyme’s active site.
Step 3: Catalysis
The enzyme catalyzes the breaking of a glycosidic bond.
Step 4: Product formation
The enzyme facilitates the formation of smaller carbohydrate compounds.
Step 5: Enzyme reuse
The enzyme is available to react with a different starch molecule.
The enzyme is not converted into the final carbohydrate product.
Why Does Starch Gelatinization Matter?
The most significant limiting factor in the performance of amylolytic enzymes is the physical state of the starch.
The structure of the starch granules is relatively organized. Enzymes may not be able to reach starch molecules within these granules.
When excess water is present, each starch molecule becomes organized when heat is applied.
During gelatinization:
- The granules absorb more water.
- The granules become larger.
- The organization of the molecules is disrupted.
- Amylose can be released from the starch.
- The starch becomes easily accessible to the enzymes.
Therefore, significant enzyme hydrolysis occurs once gelatinization is complete.
This is especially important in processes such as:
- Cooking and modification of cereals
- Liquefaction of starch
- Brewing and malting
- Production of syrups
- Fermented food products
However, damage to the enzymes is possible if too much heat is applied. Therefore, this process needs to balance the starch gelatinization and the activity of the enzymes.
How to Make Amylolytic Enzymes Work Better
Better enzyme performance is achieved by better control of the enzyme’s environment.
The most important factors are:
The most important factors are:
- Temperature
- pH
- Concentration of the enzyme
- Concentration of the substrate
- Reaction Time
- Water Availability
- Mixing
- Gelatinization of starch
- Inhibitors
- Concentration of products
- Stability of the enzyme
Temperature
Temperature affects the activity of an enzyme.
As temperature rises, molecules gain energy, and the average speed of molecular movement also increases. This results in an increased rate of enzyme-substrate interactions.
With increased temperature, the rate of enzyme action can be expected to increase and approach the maximum rate at which enzymes work best.
Exceeding the optimum temperature, however, leads to denaturation and loss of stability in the protein structure of the enzyme.
Almost all enzyme proteins do the same. The relationships among these enzymes might be described in the following way.
- Low temperature: slow activity.
- Optimum temperature: maximum activity.
- High temperature: loss of enzyme activity.
Generally, the temperature at which an enzyme is most active may be different for different preparations.
Potentials for improvement
Instead of assuming that all amylases share the same optimum temperatures, food processors should determine the prescribed temperature ranges for the commercial enzyme preparations that are specific.
Temperature should be carefully monitored, as an enzyme’s activity may be decreased by an exposure to higher temperatures, even for a relatively short time.
pH
The pH at which an enzyme is active can be important.
The chemical bonds in amino acids that are part of the enzyme and make up the active site change their ionization state with pH. This can change the size and shape of the enzyme at that site and affect the substrate binding capacity.
There is a specific pH range within which each enzyme becomes active.
For instance, one product may have the enzyme active in slightly acidic conditions, while in other products amylase may be used which is active in a different pH range.
How to enhance performance
Optimization of the pH of the enzyme processing system should be done within the recommended pH range of the processing system.
This may involve
- Monitoring the pH of the system regularly
- Addition of food grade acids or bases when needed
- Managing variability in raw materials
- Minimizing changes in pH during processing
Remember pH and temperature should be optimized together, since the best pH for a particular temperature may not be the best for a different temperature.
Concentration of Enzyme
Increasing the concentration of enzyme will often increase the rate of starch hydrolysis as long as sufficient substrate is present.
For example,
- Low enzyme concentration will yield slow starch hydrolysis
- Optimum concentration gives efficient starch hydrolysis
- Concentration beyond optimal will yield very little benefit
- Therefore, optimal enzyme concentration is required.
Amount of Substrate
The quantity of starch has an impact on enzyme activity.
- If the starch concentration is low, the adjacent active sites on adjacent enzyme molecules are able to come in contact with the starch molecules.
- When substrate concentration is low, the reaction rate is limited by the availability of active enzyme sites.
- Michaelis-Menten kinetics describes this saturation behavior of an enzyme system.
- Increasing substrate concentration is expected to increase reaction rate up to a maximum.
- Beyond this maximum reaction rate, increasing substrate concentration will not accelerate the reaction.
- viscosity
- reduced mixing
- decreased enzyme-substrate contact
- difficulties in heat transfer
- Because of this, other processing conditions need to be optimized as well.
- High viscosity in food processing can lead to poor mixing and enzyme-substrate contact.
- Water plays an important role in breaking starch bonds by hydrolysis.
- Water also facilitates starch gelatinization and molecular movement.
If water is limited,
- starch gelatinization can be hindered
- enzyme accessibility can be limited
- mixing can be more difficult
- hydrolysis of starch can be inefficient
- Processing of cereal or starchy food products must be done within the limits of diminishing returns.
- Amylolytic enzymes take time to hydrolyze starch.
If processing time is short:
- starch conversion can be incomplete
- sugars at desired concentrations cannot be achieved
- properties of the processed product can be inconsistent
- If the processing time is much longer:
- additional conversion will bring little benefit
- process time will increase
- so will costs of energy and equipment
- In such a case, undesired carbohydrate profiles start to form.
Amylolytic enzymes will hydrolyze starch within the desired time limits and retain the most desired properties of the processed product.
Instead of using the longest treatment period and assuming the ideal reaction time is the longest, the endpoint should be used to determine the ideal reaction time.
Mixing
Good mixing is essential in food processing.
- If mixing is poor,
- some starch gets too much enzyme,
- some gets too little,
- and hydrolysis becomes uneven.
As a result, product quality is poor and inconsistent.
Good mixing fosters even distribution of the enzyme with water and starch by making good contact. It also helps achieve even temperature, pH, and concentration of enzyme and substrate.
Using Multiple Enzymes Together
- Using complementary enzymes is a good strategy to improve starch conversion.
- One example is the combination of α-Amylase and Glucoamylase.
- α-Amylase converts immobilized starch to shorter dextrins.
- Glucoamylase breaks these shorter molecules into glucose.
The process is stepwise:
Starch → dextrins → glucose
α-Amylase + Pullulanase + Glucoamylase
with too much amylopectin present, this combination is even better.
The roles are:
- α-Amylase breaks internal α-1,4 bonds
- Pullulanase cleaves α-1,6 branches
- Glucoamylase liberates glucose from the resulting chains
- This shows that enzymes should be selected based on the structure of the substrate and the desired product.
- Amylolytic Enzymes in Breadmaking
- Amylases are very important in baking.
- Starch is present in wheat flour. However, yeast especially requires fermentable sugars.
- During the process of bread making, α-amylase can hydrolyze starch to provide small sugars for the yeast.
This is very beneficial.
The advantages can be:
- Better fermentation
- Better loaf volume
- Better crumb
- Better crust
- Slower staling
- More uniform and efficient dough processing
- Though the benefits can be many, excess amylase activity can lead to:
- Excess starch breakdown can result in adequate amounts of dextrins and sugars leading to:
- Sticky dough
- Gummy crumb
- Processing issues
- An issue with undesirable texture
Amylolytic Enzymes in Brewing
Amylolytic enzymes are crucial in brewing, as starch must be converted to sugar in order for brewing to be possible.
During the malting and mashing processes, starch-converting enzymes are active and lower the molecular weight of starch in order to produce sugars.
These sugars are maltose and glucose, and others.
Fermented by yeast, these become alcohol and carbon dioxide.
A simplified process is:
In malting, precise temperature control is pivotal since different enzymes exhibit varying activity ranges.
By proper temperature and process control, brewers can determine carbohydrate composition of wort and thus determine characteristics of the finished beverage.
Amylolytic Enzymes in Starch Syrup Production
Starch can be converted to syrups containing various amounts of glucose, maltose, and oligosaccharides.
A common process consists of several steps.
- Liquefaction
- Degradation of starch and subsequent reduction of syrup viscosity is done by α-amylase.
- Saccharification
- Glucoamylase converts dextrins to glucose.
- Debranching
Pullulanase stops glycogen’s branching by the breaking of α-1,6 bonds.
By varying the enzymes used and the reaction conditions, manufacturers can create carbon syrups of unmatched qualities.
Many traditional fermented foods employ microorganisms that synthesize amylolytic enzymes.
Some examples are:
- Certain types of cereal fermentations
- Some types of soy fermentations
- Some types of fermentations of roots and tubers
- Traditional fermentations of alcohol
There are many types of microorganisms that are often found in nature that synthesize enzymes that break starch down into smaller more metabolizable units.
The breakdown products of starch, sugars, serve as a food source for microorganisms.
Starch hydrolysis is an integral part of the fermentation process and is described as the transformation of starch into sugars which are broken down by microorganisms to form food products.
Enzyme Stability
A food-processing enzyme must not only have significant activity but also be stable when subjected to various conditions of the food-processing procedure.
Stability to the following should be tested:
- Temperature
- pH
- Length of time stored in a particular condition
- General processing conditions
- Other food ingredients with which it may come into contact
Industrial enzyme preparations are selected or manipulated to fit the processing conditions for which they are to be utilized.
For example, an enzyme for use in a high-temperature (time) process must remain active for a sufficient period of time at the elevated temperature.
Enzyme Immobilization
In some instances, Amylolytic enzymes can be immobilized.
Immobilization means enzymes can be attached or trapped in a material so they can be removed from the reaction mixture and potentially used again.
Immobilization can provide:
- More stable enzymes
- Reusable enzymes
- Easier separation
- Prolonged continuous operation
However, immobilization can become cost-prohibitive, and it can reduce accessibility of substrates to enzymes.
Where immobilization is concerned, it is process-specific whether it is beneficial.
Effects of Inhibitors
Some food components inhibit/interfere with the activity of enzymes.
Some possible inhibitors or interfering components include:
- Certain minerals
- Phenolic compounds
- Processing related chemicals
- Other food components
- Extreme pH values
- Excessive temperatures
The effects of any of these will be enzyme and food dependent.
For this reason, a determination of enzyme activity must be carried out using the real or actual food matrix and not a purified starch solution.
Product Inhibition
Some products of the enzyme reaction may inhibit further enzyme activity. For example, high glucose concentrations may inhibit some enzyme systems. Therefore, in the absence of total conversion, continuous or stepwise processing may allow the attainment of a high degree of conversion.
Industrial processes use or may use:
- Sequential enzymes
- Continuous reactors
- Controlled product removal
- Optimized enzyme loading
- to increase process efficiency.
Food Safety
Though highly useful, food manufacturers must verify that all enzymes used are suitable for food applications.
Transformation to food grade includes:
- Food-approved enzyme preparations
- Production under acceptable standards
- Purification
- Microbiological quality
- Allergen impact
- Correct dosage
- Compliance to regulations
- Labeling as appropriate
The enzyme source is important. Industrial enzymes are usually microbial, meaning they are produced by bacteria or fungi, and are then purified and formulated for the intended application.
Limitations
Despite the many advantages of amylolytic enzymes, there are limitations.
The limitations can be attributed to:
- Temperature
- pH
- Food composition
- Enzyme dosage
- Processing time
- Water availability
- Inhibitory substances
- Structure of starch
Another limitation is economic. Food grade enzymes can increase production costs, so the economic use of amylolytic enzymes is of the utmost importance.
Amylolytic enzymes are essential for the modern production of food and many other products due to their ability to modify the structure of starch and convert it into simpler and more useful carbohydrates.