Enzymes: Valuable Assets

By: Suanne J. Klahorst. Contributing Editor

Promising Proteases
Two Are Better
Enzyme Expertise
Future Enzymes
What is Organic?

Enzymes - the biological catalysts regulating the chemical reactions necessary to sustain life - are regarded either as problems or solutions, depending on their impact on food processing and product quality.

For the fresh-cut fruit and vegetable processors, endogenous enzymes from plant tissues are responsible for browning, adverse flavor changes and texture loss - changes that need to be avoided by blanching, chilling, freezing, acidification or sterilization. In a bakery operation, enzymes are viewed as valuable assets that make the job of turning out consistent bakery products a little easier. Historically, malt extracts - which are rich in native barley enzymes - were added to doughs to get the benefit of those enzyme activities. Today, it is common to supplement native flour enzymes with exogenous enzymes produced by microorganisms, particularly amylases, proteases and xylanases. Some of the benefits of enzymes in bakery products are consistently improved machinability, higher loaf volume, smoother crumb characteristics and longer shelf life.

Almost all food products are made from living systems, so all foods are subject to at least one, but typically dozens, of enzymatic reactions. That's why enzymes represent not only the most challenging problems, but also provide the most elegant solutions in food product and process design.

Promising proteases

Enzymes are commonly named for the substrates they catalyze, with a few exceptions. Their purpose is to enable a reaction that alters the chemical structure of that substrate, indicated by the added suffix "-ase." Enzymes that hydrolyze protein are termed proteinase, or more commonly, protease. While proteases are specific for protein, they are quite versatile in their capacity to hydrolyze a variety of protein bonds from different sources - both animal and vegetable. For each type of enzyme and each type of protein, the end-products of the reaction are different. In the case of the specialized mammalian protease called chymosin, a limited hydrolysis of milk protein is achievable for the specific purpose of forming a protein curd for cheesemaking. Other types of proteases are able to convert solid proteins from meat, fish or legumes into liquid slurries or protein hydrolysates, as in the production of savory flavors. Some proteases are effective at hydrolyzing proteins all the way to amino acids.

Although a wide variety of bacterial and fungal proteases are now available, papain still enjoys a certain popularity as a natural plant enzyme that can achieve results similar to those of the high-tech microbial proteases that threatened to make it obsolete. Papain has enjoyed a market in several industries, including brewing, where it has been used for chill-proofing beer via its ability to degrade the protein component of haze.

Papain has its limitations, however. Most papain is imported from regions of the world where a special variety of papaya fruit is grown for its protease enzyme content. Papain importers usually have very little control over the enzyme extraction and purification process. Quality can vary as a result.

Valley Research, Inc., an enzyme company based in South Bend, IN, finally eliminated two limitations of papain. "Papain has an odor reminiscent of old gym socks," explains President Art Sears. "After two years of working with papain growers in India, we developed an improved enzyme-recovery process that not only results in an enzyme product with less odor, but eliminates the use of metabisulfite as a preservative." Valley Research also is marketing another plant protease for food processing. Called "actinidin," it is extracted from kiwi fruit.

As the selection of proteases from various plant, animal and microbial sources increases, the opportunities for combining enzymes to create new food ingredients multiply. Proteases, in particular, are becoming valuable tools in the conversion of protein byproducts into high-value functional ingredients.'

Jamel S. Hamada, Ph.D., research chemist, Southern Regional Research Center, USDA's Agricultural Research Service, New Orleans, specializes in enzymatic modification of proteins for expanded food uses. He currently collaborates with Louisiana State University in the identification and development of new uses for rice bran. "Rice bran is sold for as little as 2 cents a pound, but the bran contains from 12% to 20% protein," Hamada says. "If these proteins could be recovered and solubilized, the protein value could be considerably higher." By combining two food-grade proteases from Novo Nordisk, Basvaerd, Denmark, Hamada demonstrated that he could recover proteins from rice bran and improve them to the extent that they exhibited emulsifying functionality similar to casein. He presented the results of his ongoing research at the IFT Annual Meeting in June.

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Two are better

Enzyme companies specialize in leveraging the synergistic effects of more than one enzyme to improve the functionality in food systems. One of the most complex and precise enzyme-blending challenges is the baking industry. With specific enzymes for degrading each of the components of flour, including starch, protein, and xylans (pentosans), the possibilities for combining all the activities into one product are nearly infinite. In baking, enzyme blending is as much an art as a science. Since flour composition is not consistent, enzyme blends must be fool-proof enough to consistently deliver the desired benefits, without overtreating the dough to the point that product quality or machinability is lost. The enzymes used in baking enzyme blends also must be temperature-labile enough to inactivate at baking temperatures, to avoid residual changes in the final product on the shelf.

Some enzyme combinations are targeted for a particular flour (wheat, rye, oat) or a particular finished product, such as bread or crackers. Rye and oat flours contain sticky xylan and glucan polymers that make processing troublesome without the use of xylanases or glucanases. Amylases - named for amylopectin - convert gelatinized starch to fermentable sugar, thereby improving the leavening and anti-staling properties of bread and rolls. Fungal or bacterial protease addition results in a slacker dough and a reduction in conditioning time for cracker production.

In spite of the art required, enzyme addition to baked goods has been simplified by the availability of pretested blends that are developed in the research bakeries of enzyme companies or enzyme-ingredient suppliers. The formula for these blends is always proprietary to protect the art, but for those product developers who don't have the resources or the expertise to run the extensive product trials, this is an acceptable solution. Novo Nordisk has these baking capabilities in their Franklinton, NC, facility, where they use test enzymes in baking to create a wide variety of baking products.

Greg LeFebvre, industry manager, Novo Nordisk, Franklinton, recently disclosed the fall 1998 introduction of their newest enzyme blend in the Fungamyl® product line, Super BR, an enzyme system designed to replace chemical dough conditioners, such as potassium bromate.

"During the product test market, customers reported that Fungamyl Super BR demonstrated improved bendability and wrapability in wheat tortillas, and improved loaf volume and crust in bread," LeFebvre says. "Additionally, greater porosity in English muffins has been observed in plant trials. These are all benefits that can be realized from a gluten strengthener in baking."

Bromate was a cheap and effective dough strengthener until its good reputation was lost after its degradation products were found to be carcinogenic to rats. Europe and Canada responded by banning bromates, and although legal within specified limits in the United States, California law requires a warning label if bromate is used as an additive.

Since enzymes that are inactivated in the baking process don't appear on the ingredient label, bakeries that use bromates or chemical bromate replacers, such as azodicarbonamide and iodates, can clean up their label by replacing some of the chemical additives with enzyme blends. "During the product test market, customers reported that Fungamyl Super BR created more porosity in English muffins, improved crust appearance in hearth breads, and improved bendability and wrapability in wheat tortillas," LeFebvre reports. Fungamyl is produced from the fungal organisms Aspergillus niger and Aspergillus oryzae, which have been used for several years to produce enzymes for the baking industry.

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Enzyme expertise

There are many success stories for new enzyme applications and product uses. The most encouraging fact is that many of the ideas for enzyme blends and new ways to use them originate with the enzyme user rather than the supplier. Food scientists and technicians who gather an arsenal of enzyme samples can create unique products and proprietary processes that enzyme vendors cannot, because enzyme vendors lack access to seasonal raw materials and familiarity with the customer's process.

New enzyme applications require multiple trials and determination as much as technical enzyme expertise. Mike Turner, fruit and wine industry manager, Valley Research, provides an illustration of how enzymes can be blended for maximum benefit. In the juice-processing industry of the Pacific Northwest, Turner became familiar with enzymes that are used routinely for depectinizing and clarifying fruit-juice concentrates. While employed at a firm specializing in Concord grape concentrates, Turner became frustrated with the costly and tedious process that started at the Concord grape harvest. "Single-strength Concord grape juice is traditionally held in cold rooms for several months to allow the tartrate crystals to form and settle to the bottom before concentration," Turner explains. "So, it can be months before there is any product to sell."

Turner gathered technical information from his enzyme suppliers and Oregon State University, Corvallis, to understand how to develop a shorter, more economical process for concentrating and stabilizing Concord grape juice. The new process he developed hinged on a custom blend of four complementary enzymes he added during the crushing process. The enzyme blend, a proprietary mixture of pectinase, cellulase and hemicellulase enzymes, depectinized the juice earlier in the process and reduced the viscosity enough to improve the removal of insoluble solids during the centrifugation, called "decanting." The viscosity reduction and a lower percentage of insoluble solids was enough to make concentration of the juice possible shortly after crushing.

"The tartrates formed more readily in the concentrated juice, which decreased the hold time required," Turner explains. "After tartrate removal, the juice was concentrated further to 70°Brix for shipping. When the concentrate from the new process was compared with the traditional process, it had more color and higher juice yields per ton of grapes processed. "Reducing the hold time also eliminated the chances of contamination by yeasts and bacteria that convert juice to wine or vinegar during storage," he says. "We had grape concentrate ready to sell in a month rather than several months." Turner's enzyme talent eventually landed him his current position at Valley Research. He currently shares his processing experience with other Concord grape juice processors. His enzyme blend, which is still proprietary, is now commercially available from Valley Research as Crystalzyme® Concord.

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Future enzymes

Genetic engineering has created many new opportunities for producing enzymes in food crops. Companies specializing in transgenic plant technologies are ready to exploit the vast new opportunities for enzyme production in plants. One such company was founded in 1993 by Ray Rodriguez, professor, department of molecular and cellular biology, the University of California-Davis. Rodriguez, chairman of the Board of Applied Phytologics Inc. (API), Sacramento, CA, specializes in expressing enzymes in transgenic cereal grains. "API's current focus is on rice," he explains, "which has the smallest genome of the major cereal crops and, consequently, the best-known genome of the cereal grains." The gene transformation in rice was completed 10 years ago, in 1988.

Transgenic barley, another research focus for API, was not developed until 1994, but it offers advantages because of the potential that malting operations offer for adding value by enhanced enzyme activity. Barley is routinely malted for brewing operations, a process that allows the endogenous enzymes to convert the starch to sugar for fermentation to ethanol. One proposed target is the addition of a gene coding for a heat-stable beta-glucanase in barley that could break down most of the beta-glucan during the malting process. Beta-glucans often interfere with the separation of wort and mash during the brewing process (called lautering). Eliminating beta-glucans upfront, in the malting process, would reduce the need to add enzymes later during brewing. Incidentally, API formed a business alliance with Coors Brewing Company, Golden, CO, to examine how enzymes destabilize plant allergens, but they've yet to collaborate on brewing applications.

Rodriguez points out that grains offer many advantages for the delivery of enzymes to food products because they offer low-cost production systems. Harvesting, transportation and storage are more economical with grains than with fruit and vegetable crops. For many of the cereal grains, the secrets of transformation need to be elucidated to achieve "genotype-independent transformation." Once the targeted gene has been successfully inserted into a research grain, it needs to be transferred to a commercial variety for production. When this transformation becomes routine, the development time to get the products to the market will be reduced. Since all the cereal grains share similarities in their genomes, API is positioned to apply their transgenic rice know-how to other commodity grains, such as wheat and corn, in the future. They are forming alliances with companies interested in improving grains for delivering nutritional benefits in patented food and nutraceutical applications.

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What is organic?

Although food enzymes are usually considered processing aids and, as such, are the least visible addition to food products, there are still unresolved issues for food companies that want to use genetically engineered enzymes and maintain organic standards. USDA's recent attempts to propose national standards for organic products resulted in the confirmation that food produced by modern biotechnology does not "fit current organic practices nor meet current consumer expectations about organic," according to Agriculture Secretary Dan Glickman. That would eliminate the genetically modified corn and soybeans from the organic menu, but where do recombinant enzymes fit in this scenario?

Several organic-food processors have requested verification from their suppliers that the enzymes used in their process are not "recombinant." This statement can refer to the microorganism that hasn't been modified with recombinant DNA techniques and the enzymes produced for the new DNA sequence.

A few enzyme suppliers have indicated in their literature that certain products are "not genetically modified." However, most enzyme manufacturers do not provide this information in their product literature. If pressed, they usually disclose the required information to customers in the interest of maintaining the customer relationship. The role of enzymes in organic products has been quietly debated, since the microorganisms that produce enzymes are removed before the enzymes are concentrated or granulated into final enzyme preparations, and since the enzymes are inactivated in the final food product. In addition, the quantities of enzymes used as processing aids are often very low, usually in the ppm range.

No matter which sector of the food industry is selected, there is always a need to be more aware of food enzymes and how the problems they cause in one sector might result in a solution for another sector.

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 with the California Institute of Food and Agricultural Research at the University of California, Davis.

• Photo: Pacific Tortilla Kitchen, a Division of Specialty Brands, Inc.

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