Selecting an Enzyme
May 1995 -- Applications

By: Lucy Saunders
Contributing Editor

  Enzymes serve numerous functions in food production, from maximizing juice extraction of pectin-rich fruits to extending shelf life in baked goods through slowing starch retrogradation. Yet the evaluation and selection of enzymes for a specific food processing application can be extremely time-consuming due to the sheer number of choices. Indeed, there are hundreds of commercial brands and even more variations among each class of enzymes based on their side activities.

  In nature, there are thousands of enzymes, augmented by a few dozen man-made enzymes customized through genetic engineering to suit a specific food processing requirement. According to several industry estimates, fewer than 5% of the more than 7,000 enzymes known to humankind have been commercially adapted for food use and have been officially reviewed and approved by the Food and Drug Administration.

  Enzymes with proven food applications can save time and money, but only if all the variables affecting use of the enzyme are researched and tested. To understand these variables requires knowledge of basic enzyme functions.

About enzymes

  Enzymes are the workhorses of nature, harnessed to a substrate to catalyze certain chemical reactions without extremes in temperature, energy or additional chemicals that may produce unwanted byproducts. As long chains of amino acids held together by peptide bonds, enzymes are defined first as proteins, but their immense value lies in their catalytic actions and side actions.

  Most enzymes are implemented as Generally Recognized as Safe (GRAS) additions to the food production cycle. Since the enzymatic changes occur at the cellular level, enzymes are viewed as functional aids to processing, not as ingredients. Enzyme activity can be targeted to the specific goal of the food processor, without requiring what Tom Meyaard, marketing manager of Solvay Enzymes, Inc., Elkhart, IN, describes as the "fire and brimstone" of other chemical or temperature processes.

  "Look at the basic nature of an enzyme," says Meyaard. "It serves as a catalyst that will trigger a specific chemical reaction and, in general, it will accomplish that chemical reaction in an easier way than some other process. Enzymes typically operate at lower temperatures and at a lower pH than the corresponding chemical reaction, so researching an enzyme should be the first response when trying to find an easier, less expensive method to accomplish a reaction."

  Meyaard points out that enzyme selection is complicated by the fact that it tends to be more of a problem-solving application, rather than an ingredient that is specified for the formula from the inception of the product design. "The product designer may have to consider the enzyme late in the development cycle, as a means of troubleshooting," Meyaard cautions.

  Given that there are hundreds of enzymes generally recognized as safe (GRAS) for food use, the selection process should be well targeted to speed the evaluation process.

  "It is important to define both what you want the enzyme to perform as its principal reaction and what cannot be tolerated as a side activity," says Meyaard. These two opposites will determine the parameters for the search.

  Fortunately, enzymes are highly specific in the primary actions -- so much so that it is possible to obtain enzymes that act only on certain molecular bonds in the substrate compound, limiting the number of byproducts that would then have to be removed from the finished product. Meyaard cites the example of the soft drink industry, which uses vast amounts of high fructose corn syrup. To meet their needs for bulk HFCS, enzymologists developed low-cost, genetically engineered enzymes to speed syrup production. Of course, HFCS can be produced without enzymes, but the syrup supplier then winds up with tremendous costs in terms of energy and additional acid chemical processing to remove the degradation byproducts.

  "It is so much easier to make HFCS with an enzyme such as alpha amylase, glucoamylase or glucose isomerase that works without creating any other strange byproducts," comments Meyaard.

  Furthermore, enzymes may be blended for more efficient catalytic reactions. Again using the example of syrup production, processors may choose from blends of glucoamylase and pullulanase for more efficient starch saccharification systems with shorter hold times and higher glucose yields. The pullulanase will hydrolyze specific glucosidic branch points in starch for faster conversion to glucose by glucoamylase. Or, in fruit juice production, a multienzyme-derived cellulase will convert soluble cellulose derivatives and native cellulose, speeding extraction and clarification of pulpy citrus fruits.

  Because so many applications for enzymes exist in baking, brewing, distilling, cheese-making and dairy products, flavor systems, fruit juice processing, syrup and starch systems, finding the proper enzyme can be challenging. For example, just one class of enzyme -- the lipases -- can be used in many ways: in producing monoglycerides for emulsion applications in cosmetics; in modifying fats such as palm oil with stearic acid through interesterification for use in chocolate confectionery; in developing savory flavors through fat hydrolysis; and in speeding the aging of cheeses.

  "Cheese-makers now have a choice in making cheeses and aging them for four to six months, or using an enzyme and shortening the length of time required to develop the nutty flavors of aged cheese," says Meyaard.

  The wide range of applications for the sole class of lipases points to both the versatility and variability of enzyme activity.

  "Start by looking at a relatively large picture and then narrow it down," suggests Greg Le Febvre, market development manager for Novo Nordisk Bioindustrials, Inc., Danbury, CT. "Determine what you want to achieve: Do you want to reduce viscosity? Create a change in functionality of the raw materials? Change a flavor characteristic? Those are the types of questions that a researcher has to ask when selecting and evaluating an enzyme."

  Le Febvre cautions that enzymes are appropriate for some tasks and not for others, depending on the substrate. "The three classic divisions for enzyme applications are based upon the substrate system: Is it a starch, a protein or a fat? However, the increased use of other enzymes, such as oxidases that remove oxygen from a system, points to other food processing possibilities."

  Other factors to consider when selecting an enzyme are the general conditions for your food or beverage product, such as processing parameters of temperature, pH, water content, reaction times necessary and other operational factors. It is critical to test the enzyme activity under the real conditions of your plant, since laboratory tests alone may not indicate the changes in enzyme activity due to even slight fluctuations in temperature or pH.

  Enzymes derived from fungal sources typically are most effective in slightly acid conditions (pH 4.5), while other enzymes from bacterial, yeast and animal sources are more effective under slightly alkaline conditions. Heat-stable enzymes are available for many applications, but they may cause protracted enzymatic activity that ultimately degrades the shelf life or consistency of the food product.

  Most enzyme producers try to characterize the enzyme products extensively through assays. The vendors need to know the primary activity of the enzyme and all the possible side activities. For example, many of the fungal protease enzymes used in flour production also have significant amylase activity, as well. Or, a protease typically used as a partial malt replacement in brewing also might exhibit gluconase activity.

  With proper testing, an enzyme company can define what specific chemical modification is required and correlate it to the range of enzyme activities. Through genetic modification, more enzyme companies will soon offer mono-component enzymes, which have only one catalytic activity with a very high degree of specificity. Eventually, food processors will be able to create highly targeted modifications of a substrate to achieve a particular processing goal.

  Although the numbers of commercially available enzymes now reaches into the hundreds, there are few standardized sources for enzyme assays that can be compared across brands and suppliers.

Digging up data on enzymes

  R&D departments face a challenge in evaluating enzyme activity. Due to the great range of applications and very different biological sources for enzymes, there are numerous factors to consider when weighing the enzyme assays provided by a manufacturer. Moreover, many suppliers use different scales in measuring enzyme activity. In fact, it sometimes makes good economic sense to hire a consulting enzymologist for the evaluation, simply because the subtle differences in assay results when compared across brand lines can translate into major differences in processing. That is because enzymes are so powerful, even in small amounts, that the most minute variations in dosage or dilution can wreak havoc. For example, in baked products, it is possible for alpha-amylase enzyme activity to continue even after baking, creating a gummy texture or crumb in the product. Hence, an accurate enzyme dosage at the outset is important. Enzymes already present in certain flours, syrups and other bakery ingredients also should be factored into the equation to determine overall enzyme activity.

  Variations in measuring enzyme activity can be confusing. When a product designer is trying to compare activity assays of a particular class of enzymes across brand lines in order to estimate quantities to be used and their associated costs, knowing the basis for the scale of enzyme activity measurement is critical. The amylose activity of malt extracts and syrups is usually expressed in degrees Lintner, while the activities of more concentrated forms of amylase are typically expressed in Sandstedt-Kneen-Blish (SKB),units. Slight variations upon the SKB method can be found in the assays reported by different producers of amylase, which can only create further confusion.

  A trained enzymologist can assist in the evaluation of several different sources for the same enzyme so that you can determine if your supplier tends to err on the side of overestimating or underestimating activity levels. In some cases, suppliers underestimate activity levels to compensate for the possible loss of activity during enzyme transport and warehousing.

  Knowing if there are any differences between the quoted ranges of activity and your laboratory-tested range of activity can save substantially on your production costs. Naturally, the more highly purified the enzyme is regarding its activity, the more expensive it will be. Understanding the limits of tolerance for side activities of a given enzyme can save money, as well, by enabling you to purchase the crudest, but also least expensive, enzyme that will work in your application.

  Testing an enzyme in your own facility is necessary to gauge any effects from your equipment and production process. "There are some enzyme suppliers who work hand-in-hand with equipment manufacturers to ensure that the enzymes used do not interfere with the operations of the food or beverage processing equipment," says Novo Nordisk's Le Febvre.

Collaboration and change

  Collaborative efforts between enzyme suppliers and equipment manufacturers in the fruit processing industries have yielded good results. Some of the enzymes used in fruit juice production for enhanced juice clarity or to improve the juice yield have been optimized for use with specific types of equipment, such as centrifuges or fruit presses. This kind of collaboration among a fruit juice processor, an equipment producer and an enzyme company can maximize the investment to obtain the best yields possible.

  Though slow and painstaking, such collaborative efforts are worth the time. As Meyaard says, "The whole industry only works -- when there is good communication between a client and customer. It's a slow process to develop both the product and the equipment, but the enzyme suppliers have many applications for enzymes as yet not commercialized that could be brought to bear on a specific food processing project."

  In one example, a blended starch debranching enzyme took 15 years to develop in tandem with a large wet milling operation. The new enzyme blend operates at a pH more suited to a large-scale producer of HFCS. Despite the costs involved in producing the new enzyme, ultimately it has saved the HFCS processor quite a bit of time and money, creating a cleaner product without additional costs. for pH adjustment and ion exchange operations.

  More enzymes are being developed to treat waste products. In the meat packaging industry, there are vast quantities of scraps, once relegated to the renderer, that now may be treated with enzymes to produce meat flavor ingredients suitable for the pet food industry. As the desire for an all natural label extends even to pet food, the alternate sources of protein from scraps can be used to create value-added products.

  Hydrolyzed vegetable proteins, such as soy, can be made more digestible and better tasting as a nutrient boost for soups, sauces and prepared foods. Enzymes also can be used to create modified wheat gluten for vegetarian food products.

  Even caseins and whey proteins can be modified with an enzyme blend to create savory flavors for the cheese industry or to enhance yogurt and other milk-based products. In the dairy case, some of the greatest experimentation with enzymes occurs with the use of lactase to reduce the lactose content of dairy foods, modifying the products for the lactose-intolerant market. As baby boomers continue to age, the need for lactose-reduced dairy products will continue, spurring even more growth in enzyme-modified milk products. Even a dairy-based sports nutrient replacement beverage is being created from whey protein through the use of enzymes.

  The more highly targeted enzyme blends can still be affected by activators or inhibitors masquerading as ingredients or nutrients. Some enzymes require minerals, such as calcium, to perform; any other ingredients in the product formula, such as ethylene-diamine-tetra-acetate (EDTA), would interfere with such enzyme activity by chelating the available calcium. Differences between the enzymes in a blend -- such as different inactivation temperatures, varying pH requirements, or other conflicting factors -- should be tested and evaluated in the entire product formulation.

Advances in handling

  In the baking industry, enzymes are useful for extending shelf life and product quality, yet the method of dispensing them is critical. Though new enzyme formulations are specifically designed to prevent overdosing, the need for easy-to-measure forms is even greater with the widespread use of automation on the bakery floor. Tablets are often preferred over powders, both for purposes of accurate measurement and safety in handling. Again, since enzymes are so concentrated and are usually added to bakery products at a rate of less than 0.5% of total flour weight, the pre-measured tablets are much more convenient to use.

  Powders, though traditionally considered the more stable form for an enzyme, are more difficult to handle without waste or risk of overdosing. Dilute liquid enzymes are extremely easy to measure, but the more dilute the solution is, the greater the risk for a microbial contamination that could inhibit enzyme activity.

  Joe Sigel, Danisco Ingredients, Grindsted Division, Industrial Airport, KS, points out that tablets are simpler to handle since they are easily miscible when blended with a small portion of water and added to the sponge or dough. Of course, enzyme tablets must be completely dissolved for even distribution throughout the dough. Dough-strengthening enzymes are not heat stable, and at 170°F the enzymes will lose their functionality. By adding the dough strengthening enzymes to the sponge, the enzymes will catalyze during the yeast fermentation cycle and finish their activity at the time of baking.

  As the use of bromate has been voluntarily discontinued by the baking industry, enzymes assist in replacing its functionality. Working in tandem with oxidants such as ascorbic acid, the alpha amylase enzymes can be used as a dough strengthener in lieu of potassium bromate. However, allowing sufficient time for the desired level of enzyme activity is another important parameter to test. Even the best mixed tablets and powders will not achieve the desired effect if the reaction time is cut short.

Sourcing enzymes

  Reference books, such as "Enzyme Nomenclature," published by the International Union of Biochemistry, make a good beginner's reference for enzyme selection. "Enzyme Nomenclature" offers enzyme descriptions, listing sources and reactions, as well as the chemical modification that the enzyme will perform.

  Enzyme suppliers publish catalogs of research-grade enzymes for testing. While the list of commercially available food enzymes is somewhat limited, there is a huge sample base of enzymes for research uses. These enzymes can then be adapted by a commercial provider for actual use and scaleup. Of course, the enzyme food approval regulatory process could cost the producer more than $500,000 and require substantial time for governmental review. The financial incentive must be sufficient to cover the initial investment just for the regulatory expense, with each new idea for an enzyme product screened according to its economic viability.

  Designers should work with several suppliers at once when conducting the initial review of enzymes for a food processing application. Evaluate different samples of each company's appropriate enzymes, and compare the ranges in minimum activity levels. Test each batch of samples; do not rely only on the provided assay information, since other factors may influence enzyme activity in your product's formula.

  A consulting enzymologist also can speed selection and evaluation of an enzyme. Since it makes good economic sense to pre-screen the enzymes required, it also makes good economic sense to hire an independent laboratory that specializes in enzymes to assist in the research phase and to clarify data in the enzyme evaluation process.

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