Designer Enzymes Create
New Forms and Functions
March 1997 -- Applications
By: Suanne J. Klahorst
Contributing Editor
Adding enzymes to foods to create new and unique products dates back to traditional cheese-making practices. Since discovering that stomach enzymes curdle milk, humans have extracted protein-modifying enzyme mixtures called "rennet" from unweaned calf stomachs for coagulating many popular cheeses.
As calf-stomach supplies dwindled, enzyme biotechnology was utilized to develop a rennet alternative. This took the form of chymosin, which is virtually identical to calf enzyme except it is produced by a microbial fermentation process. The cheese industry's acceptance of new chymosin enzymes makes it one of the most successful applications of food enzyme biotechnology.
The new fermentation chymosins have captured more than 80% of the animal-based rennet market because of its consistent functionality, flexibility and value, says John Lippman, market development manager with Chr. Hansen Inc., Milwaukee.
Technologies similar to those that created the new chymosins have expanded selection of many other new enzyme products targeted for the food industry. Utilizing today's vast array of food enzymes requires basic knowledge of their various forms and functions, and staying current on new enzyme technologies responsible for making them available.
Form follows function
As nature's biological catalysts, enzymes enable chemical reactions to proceed within life-sustaining temperature and pH ranges. They are named after the substrate of the chemical reaction they catalyze by adding the suffix "-ase." For example, proteases modify protein; glucanases hydrolyze glucans; and glucose oxidase oxidizes glucose.
Enzymes are protein molecules folded into three-dimensional structures enabling momentary chemical binding to a substrate. Once the enzyme is chemically bound to the substrate, the enzyme protein can alter the chemical energy required to catalyze a specific reaction at the enzyme's active site.
Enzymes are generally referred to as specific, because their structure only allows them to catalyze one type of chemical reaction for one type of substrate, such as hydrolysis, oxidation or reduction. Enzyme proteins are similar to food proteins in that enzyme protein structure is disrupted by changes in temperature, pH, ionic strength and other physical factors. Once an enzyme's structure is irreversibly altered (denatured), the enzyme is considered to be inactivated and loses its catalytic function or activity.
Most enzymes catalyze reactions within the temperature and pH ranges of the organism from which they are derived. For enzymes from a thermophilic bacteria such as Bacillus stearothermophilus, the functional temperature range can extend to the boiling point of water. For a fungal, plant or animal source, it can range from ambient room temperature to 140(F).
In general, the higher the enzymatic reaction temperature, the faster the reaction will proceed until the point at which the enzyme is inactivated by heat, pH or combinations of several factors. Heat-stable enzymes from heat-tolerant bacterial sources are preferred for degrading gelatinized starch in sugar-syrup production. However, when temperature and pH changes are used to control or inactivate enzyme activity in a food process, stability to heat and pH also can prove disadvantageous.
Using temperature and pH to control enzyme-reaction rate is a valuable application tool for food product design. New enzyme products being introduced to the food industry take advantage of these principles to meet specialized needs for food-processing niches. For example, the high temperature amylases being used for sweetener-product production are no longer the preferred enzyme for driving a similar starch degradation reaction in a baked good.
Case study
Practical knowledge of temperature and pH control of enzyme activity are fundamental to using enzymes designed for the baking industry. One example of a food enzyme developed to meet a particular industry's specialized need is Novamyl(, a second-generation bacterial (-amylase enzyme offered by Novo Nordisk of Franklinton, NC. This enzyme has been widely accepted as a processing aid to retard staling of bakery products.
Staling retardation is accomplished by the enzymatic conversion of starch to sugars and dextrins of primarily degree of polymerization 2 to degree of polymerization 5. This conversion improves water retention of baked goods while reducing retrogradation, the chemical process that causes staling in baked goods.
Earlier (-amylases were responsible for a dough defect called "gumminess" if not properly controlled. As a result, baking experts developed a preference for more heat-labile fungal (-amylases. The fungal amylases modified less of the starch, sometimes only 10% to 12%, before they were readily inactivated in the oven. Hence, gumminess was eliminated. But starch which gelatinized after the enzyme was inactivated remained intact to participate in the staling process.
"Even when tested in baked goods at 10 times the recommended level, this bacterial (-amylase will not create a gummy dough," says Todd Forman, senior chemist with Novo Nordisk technical service, " because the end products are sugars and dextrins with a lower molecular weight than produced by the earlier (-amylases. Novoamyl (second generation alpha amylase) has a lower temperature of inactivation than first generation products. It has intermediate thermostability between that of traditional bacterial alpha amylases and fungal alpha amylases."
Drawing on curves
Determining a food product's compatibility with enzyme temperature and pH requirements usually begins with the temperature and pH curves provided by the enzyme supplier. These curves are offered as general guidelines and are not always indicative of how the enzyme will function in a food product. The data provided by these curves is typically generated by enzyme activity assays on artificial substrates in buffered solutions.
As an example, the pH/relative activity curve for Novo Nordisk's Novamyl indicates that this enzyme would not be expected to perform well in a sourdough bread below pH 4.0, since the curve shows only 25% of the potential enzyme activity at this pH. Good results might be possible in an actual bake test, however, even though the curve indicates it would require four times the recommended enzyme level. This is possible because enzymes might exhibit enhanced stability within the matrix of a food product that they do not exhibit in a test-tube assay. The opposite also can occur: The food product might contain an enzyme inhibitor that prevents anticipated activity.
Different temperature and pH combinations are known to act synergistically to inactivate enzymes. Temperature and pH curves simultaneously describing both variables are rare, such as optimum temperatures for enzyme activity over a range of pH. If available, these two variable curves would illustrate that the optimum temperature for activity at pH 5.0 might not be the optimum temperature for pH 4.0 or pH 7.0.
Temperature and pH interactions can be used to an advantage. If an enzyme must be used outside the optimum pH range, lowering the temperature can improve the enzyme stability. When inactivation of an enzyme is required in a heat-sensitive product, as in the case of protein foods such as eggs or seafood, acidification can be used to inactivate enzymes with heat treatment.
In all cases, observing enzyme action in the final product is almost always the preferred method for determining the extent that a particular enzyme will be affected by the combination of these variables.
Endo- and exoMost
Commercial enzyme preparations contain a mixture of different enzyme proteins with different activities on the same substrate. Each of these individual enzymes can be useful in production of food products. Hydrolases are the most common enzyme classification used for food processing, and are responsible for clipping large molecules into smaller ones.
Hydrolases can be grouped according to specificity by the prefixes endo- and exo-. The prefix refers to the point at which the enzyme clips the polymer: endo- for middle and exo- for end.
Endo-activities are particularly useful for reducing viscosity caused by long polymers such as proteins, pectins, gums and other naturally occurring polysaccharides that cause problems in production of clarified beverages, doughs, grains, and fruit and vegetable products.
Exo-activities are useful for producing predominantly small molecules from polymers (some of which are reduced by "endos" first) such as mono- or disaccharides, dextrins, amino acids or peptides.
An example of an application for exo-activity in grain processing is the use of barley (-amylase, an enzyme extracted from raw barley grain. It is used for conversion of liquefied starch to high-maltose syrup from corn, potato or wheat starch. Barley (-amylase releases maltose units by hydrolyzing the 1,4-(-D-glucosidic linkage from the nonreducing end of the dextrin chain, a reaction which enables a conversion of liquefied starch to syrup containing up to 60% maltose. Unlike (-amylase and glucoamylase activity, it can be used to produce maltose exclusively, without additional glucose formation.
Most first-generation enzyme preparations consisted of enzyme mixtures because the production organisms used in fermentations typically make several types of enzymes for survival. Through use of biotechnology, fermentation process technology, and new protein purification techniques, several new enzyme preparations are being designed for their ability to deliver only enzyme activities that contribute to food-product enhancement.
Pure enzyme
An enzyme preparation's "purity" refers to the number of different enzyme proteins in the preparation mixture. (This shouldn't be confused with the "purity" implied by use of food grade manufacturing practices.)
In addition to the primary activity stated by the manufacturer, other enzyme activities called "side activities" might be inherent to the production organism used for enzyme manufacture. Enzyme side activities can be beneficial, inconsequential or deleterious since foods offer several potential enzyme substrates, including proteins, carbohydrates and lipids. The level of side activities can be influenced by the source. Enzyme production can use several sources, most commonly plants, animals or microorganisms, Some sources result in more side activities than others.
Most enzyme suppliers offer products that have been purified to remove undesirable enzyme activities. For example, glucose oxidases remain active in a food product in order to scavenge oxygen to prevent oxidation. They might not be suitable for use in salad dressings if they contain low levels of side activities that slowly hydrolyze the polymers used for building viscosity. This type of application requires a purified glucose oxidase.
"(Demand exists for a) wide variety of glucose oxidase products that can meet specific needs for purity depending on the end-use," says Chris Penet, technical services manager at Genencor International, Inc., Rochester, NY. Looking for potential side activities allows food developers to eliminate surprises in the product development process.
Enzyme suppliers typically provide information about side activities in their enzyme preparations when requested, and sometimes feature them as selling points in their product brochures. Within the product development process, long-term product-stability testing can detect enzyme side activities in addition to determining proper enzyme inactivation parameters.
Purification techniques
The fastest-growing group of new enzyme products is typically manufactured by a fermentation process using one of a few, FDA-friendly, nonpathogenic (such as Aspergillus, Bacillus or Trichoderma).
Purifying enzymes from microbial fermentations can be done before, during or after fermentation. Organisms for enzyme production were traditionally manipulated before fermentation by random mutation and individual selection for predominant activities. Now, biotechnology tools provide the means to delete genes that code for objectionable enzymes. Enzyme biotechnology companies also are able to move genes that code for enzymes from wild-type organisms into more suitable, food-grade production organisms.
In the fermentation process, sterile conditions prevent contamination from other microorganisms that may produce offending side activities. After fermentation, once the recovery process is complete and all the production organisms have been removed, columns for protein separation can be used to remove offending activities and further purify the enzyme.
The purified enzymes are referred to as enzyme components in some cases, since they were once a component of a larger enzyme system or enzyme mixture. Once purified, components can be sold separately if use for them exists, or sold as part of a unique enzyme blend targeted toward a particular industry's specific need.
Improving enzyme forms
Liquid food products generally incorporate liquid enzymes, and dry foods generally require dry enzymes. This is primarily a matter of convenience, but there are some other considerations to be aware of when testing enzymes in the laboratory and food production scale-up.
Liquids can be less costly as a rule, but have a shorter shelf life, and contain more preservatives and stabilizers. Enzyme protein can also serve as a nutrient that can support microbial growth. Therefore, sodium benzoate, methyl parabens, potassium chloride or sodium chloride are added to extend shelf life. Most food-industry liquid enzymes are kept refrigerated, and have expiration dates of up to one year. They can often be kept longer if stored properly.
Dry enzymes are available in several forms, including spray-dried powders and many types of granules. Preparations that have minimal water content have a longer shelf life, and might contain fewer stabilizers and preservatives.
Depending on how powders and granules are manufactured, they also can be more costly due to the extra manufacturing steps required. However, shipping costs can be less since the product contains less water and does not require refrigeration during transport.
The trend toward benzoate-free liquid enzymes appears to be continuing in several industries, particularly fruit-processing. Scott Brix, sales and marketing director of Valley Research, Inc., South Bend, IN, emphasizes the importance of providing preservative-free enzyme preparations for use in products destined for Japanese markets.
"The level of detection for sodium benzoate is in the range of parts per billion," Brix says, "and more customers are requesting benzoate-free enzymes for food products exported to Asia and foods developed for the natural foods industry."
In some cases, enzyme stabilizers such as sorbitol, glycerol or propylene glycol, also are unacceptable to some fruit processors. For these customers, dry enzymes might be more suitable. For high-volume enzyme buyers, custom formulations sometimes can be ordered from suppliers to meet special requirements.
Enzymes have to be handled using precautions to avoid inhalation. Spray-dried enzyme protein is sometimes "de-dusted" using various methods. Safety guidelines for handling liquid and solid enzymes can be obtained from the enzyme supplier or from the Enzyme Technical Association, Washington, DC.
The newest enzyme technologies are emerging in the form of granulated enzymes, which offer the advantages of enhanced handling characteristics by their association with a carrier in which the enzyme protein is applied or encapsulated. This carrier usually is designed to be compatible for the intended food application since it becomes part of the final food product. The trend in the enzyme industry is to offer new types of granules that release enzyme activity at the proper time, while protecting enzymes during storage and handling.
Future outlook
Enzymes are important food ingredients and processing aids, and second- and third-generation enzyme products will continue to be developed for the food industry.
As nutraceuticals gain popularity, enzymes that offer benefits for digestion, such as lactase and (- galactosidase (an anti-flatulence enzyme), will gain popularity. Other types of food-grade enzymes claiming to impart digestive benefits to humans have appeared on the market, but more clinical testing is needed to quantify the benefits. For the ruminant stomach, using various digestive enzymes is well-established in the animal-feed industry.
For many enzymes that have generally-recognized-as-safe status, enzymes are used as processing aids and are inactivated before consumption in a food product.
New cutting-edge enzyme technology involves the use of enzymes for forming as well as cleaving bonds. For example, the enzyme transglutaminase has been introduced in Europe for cross-linking peptides in proteins. The new enzyme is designed to offer thickening in protein-based foods without using heat.
Use of lipase enzymes for synthesis of esters is another application that could find a way into the food industry. Lipases also are being explored for modification of fats using chemical processes called transesterification or interesterification. The new enzymatic process utilizes lipase enzymes to rearrange or replace specific fatty acids within triglycerides to improve their properties.
Other trends are surfacing. Purified enzyme components and new granulation and liquid technologies suitable for delivery of enzymes into food products will dominate the new enzyme product introductions. Uses of specially designed blends of endo- and exo-activities that achieve desirable effects will continue to be developed and patented. In the protein hydrolysate industries -- which produce savory flavors from yeast, soy, fish and meat protein hydrolysis by using protease enzymes -- the trend will be to design effective enzymes that don't cause bitter flavors.
Enzyme companies will continue becoming more specialized and savvy about the food industry in order to design enzymes that solve specific problems. They are establishing their own food-product development labs and hiring food-processing specialists to manage their enzyme-product testing laboratories.
Natural approach
Enzyme biotechnology companies are taking a more conservative approach to developing new food enzymes than in nonfood industries due to regulatory-approval process obstacles , long product development cycles, and a reluctance by consumer-wary food processors to adopt biotechnology-based enzymes. Enzyme companies that have programs for discovering new enzymes from unique organisms in natural habitats, such as the heat-resistant varieties found in the thermal hot springs of Yellowstone National Park, usually don't have the food industry in mind as they are collecting samples.
Although production organisms used for food-enzyme fermentation might be genetically manipulated, enzymes obtained from these organisms are generally not. Additional clarification is needed by food companies struggling to interpret how enzymes meet new biotechnology guidelines being established by groups such as the National Organic Standards Board in response to bioengineered food crops entering the market.
Enzyme biotechnology products are viewed favorably by consumers, especially when enzymes replace chemicals considered harmful, such as bromates.
The companies that will be actively designing truly unique products for the food industry might continue to decrease. A rash of mergers and acquisitions has reduced the number of large companies that have new enzyme technology adaptable to food use. This will make room for smaller, specialty enzyme companies, some of which market to only one or two related industries, such as dairy or juice processing.
Companies also are experimenting with new ways to get the word out on enzyme products. Donald Krull, vice president of business operations for the food group of Novo Nordisk, estimates their new web site featuring product information in the United States will be on-line early this year.
Several enzyme companies offer product lists and contact information from the Internet. Product developers who understand basic principles of utilizing enzymes to create new and unique food products are more likely than ever to find a solution for their problem from the expanding selection of novel food enzymes.
Suanne Klahorst is a free-lance food-science writer and food scientist with eight years of experience in the industrial enzyme industry. She is currently employed by the California Institute of Food and Agricultural Research at the University of California, Davis.
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