Breaking Down Enzyme Formulation

For non-living organisms, enzymes sure do a lot of work. In the body, they help break down compounds, importantly nutrients. It isn't easy sorting out the right enzymes for the right applications; but, breaking down how each category of enzymes works and how each can function in modern natural product formulations is a good place to start. Still, there are many other considerations formulators must make, including safety, clinical research and the many challenges the chemical properties of various enzymes can present in production.

Enzymes are globular proteins that act as catalysts for specific chemical reactions, which means they break down compounds (substrate) to smaller compounds. There are more than 3,000 types of natural enzymes identified by scientists. Why so many? Each enzyme catalyzes only one type of substrate. This specificity is useful in many fields, including various chemical, pharmaceutical and nutraceutical industries.

Enzymes are not living organisms but come from animals, plants, fungus or microbes. These living sources utilize enzymes to break down nutrients in food, or fuel. As catalysts of the breakdown of nutrients, food enzymes are considered hydrolases, because they speed up the reaction of these compounds in water. Hydrolase enzymes can be further categorized as carbohydrases (such as amylases), proteases, lipases, cellulases, pectinases, xylanases, hemicellulases and other subtypes.

One of the simplest ways to understand the how enzymes catalyze specific substrates is to imagine them in a lock and key model. Enzymes have specific binding site shapes that fit a certain substrate in near-perfect fashion, like a key in a lock. Once bound, the compound is "unlocked" by the enzyme, separating the compound into smaller pieces. In reality, the binding is not perfectly rigid, and the enzyme can change shape slightly toward a perfect fit with the substrate.

These mechanisms show why enzymes are a dietary necessity. The primary enzymes active in nutrition, along with the substrates they catalyze, are: proteases (proteins), amylases (carbohydrates), lipases (fats or lipids) and cellulases (cellulose fibers).

While many enzymes needed for digesting and breaking down nutrients for absorption are made within the body, many also come into the body with the foods they are eventually to digest and help metabolize. This seemingly intelligent design of nature has a few obstacles, at least in modern lifestyles. Over-processing foods can denature the enzymes natural to those foods, whether plant or animal. Thus, these enzymes would not be available to help the digestive system break down the important nutrients.

Enzymes also can be vulnerable to various types of inhibition, competitive and non-competitive, which sometimes occurs with drug compounds. In certain cases, a drug compound can have the same shape as the intended substrate and, thus, compete for binding on the enzyme. In some cases, the binding is irreversible, and the enzyme is rendered inactive. Due to these and other factors, supplementing the diet with food enzymes has become a popular method of improving nutrient absorption from the modern diet.

While digestion and nutritional support are the obvious markets for enzymes, the greater area of natural health maintenance has found condition-specific benefits from the use of systemic enzymes. This category of health-crusaders is predominantly comprised of certain types of proteases, such as bromelain from pineapple, papain from papaya, and serrapeptidase and nattokinase from bacteria. As catalysts of proteins, systemic enzymes affect various proteins involved in the immune response and the inflammation cascade.

In the immune system, enzymes help deal with antigens, invaders such as viruses or bacteria that can trigger illness, injury or disease. Antibodies attach to antigens and form immune complexes, which can be broken down into benign fragments by certain enzymes. This cleaving action is effective whether the immune complexes are newly formed or have escaped initial destruction by macrophages and set up shop in body tissues and organs, ready to cause autoimmune disease. Systemic enzyme therapy has been shown to improve the body's defense against these antigen immune complexes and their downstream effects. Research is increasingly confirming hydrolytic enzymes should be part of the therapies for diseases marked by high levels of circulating immune complexes (CICs), including AIDS/HIV.

In other areas of the immune system, tumor development and progression can be inhibited by systemic enzymes. Enzymes can break down the fibrin coatings on cancer cells, bringing them out of hiding, so that immune cells can more efficiently and quickly eliminate the cancer. In addition, recent work shows enzymes may also help slow the metastasis or migration of tumors.

A century ago, scientists made the connection between cancer-fighting enzymes made in the human pancreas (where endogenous enzymes are made for digestion) and the potential for cancer therapy from enzymes derived from the pancreas of another animal, namely the pig. Supplemental enzymes sourced from animal pancreas include trypsin and chymotrypsin. In the realm of cancer, papain from papaya is also added to proteolytic blends for adjunctive therapy against cancer.

Another key aspect of immune health affected by enzyme activities is inflammation. One of the newer areas of research on enzymes is in inflammation or, more specifically, diseases and conditions marked by inflammation, which appear to improve with administration of systemic enzymes. While research into the exact mechanisms of enzymes in this area of immune health is still in early stages, scientists have found systemic enzymes promote healing by managing the inflammation process, as opposed to a blanket inhibition of inflammation, which is the way conventional anti-inflammatories work.

Proteolytic enzymes tend to congregate at sites undergoing inflammation. They help control levels of various protein compounds (peptides) involved in inflammation, including various cytokines like TNFalpha. The hypothesis is these enzymatic actions prevent chronic or harmful inflammation without the side effects of conventional therapies. The excitement here is the potential for application of this benefit in inflammatory disease in the joints (arthritis), lungs (asthma) and the cardiovascular system. In fact, recent research has found bromelain can attenuate inflammation in asthma, ulcerative colitis and post-surgical healing.

In addition, the fibrin-clearing action of enzymes such as bromelain, serrapeptidase and nattokinase have proven crucial to inhibiting atherosclerotic developments in the vascular system, including the arterial walls.

Mike Smith, sales and marketing manager for Specialty Enzymes, called systemic enzymes "an exciting area of investigation." He reported while bromelain is among the oldest and most well-known systemic enzymes, newer protease enzymes include nattokinase, serrapeptidase and blended combinations that employ one or both of these with other proteases. "These two enzymes, in particular, exhibit profound fibrinolytic as well as anti-inflammatory activity," he said. "The potential for use in inflammatory and cardiovascular conditions is significant."

Overcoming Challenges in Enzyme Delivery

While digestive enzyme products do not need to be absorbed to fulfill their purposes, systemic enzymes need to reach the bloodstream to perform their tasks. However, enzymes are sensitive to heat and pH levels, which presents a problem in the harsh environment of the gut. Therefore, systemic enzymes need to be specially coated to survive the digestive system and be absorbed into the bloodstream. "Enteric coating is used for certain enzymes to pass through stomach acids to be broken down more readily in the intestines and the pancreas," said Brett Wyant, sales, American Laboratories.

Surviving the stomach is also a concern for certain digestive products, in so far as some target areas farther down the gastrointestinal (GI) tract. For these products, newer sustained-release and controlled-release delivery technologies may be suitable solutions.

Interestingly, not only can these enzymes improve the digestion of food, but they can also reduce stress in the GI tract, help maintain normal pH levels and promote the growth of healthy intestinal flora.

New approaches and technologies are also keys to taking enzymes beyond dietary supplements to functional foods and beverages. Enzymes used to help make food products are typically not functional past their production benefit. The life of an activated enzyme can be a handful of minutes or several weeks; they are each different in duration of usefulness.

"Enzyme stability will always be the controlling factor in how enzymes are used—temperature, pH, moisture and light can all affect enzyme activity," Smith noted. For example, enzymes are not suitable for a premixed beverage, as liquid activates the enzymes to start their work prematurely. "Over a relatively short time they begin to lose their activity, becoming simply inactive proteins," he explained. "Therefore, the typical shelf life required for a premixed beverage is too short to be practical."

One way to include enzymes in beverages is to add them to dry powders that can be sold as ready-to-mix drinks, such as protein shakes. Since the water is added only when the drink is to be consumed, the enzymes would be activated at the appropriate time. Effervescent products containing enzymes could work in much the same way.

Many processed and functional food products involve heat and moisture, which can destroy the enzymes or cause them to begin their catalyzing activities. "Anything that is pasteurized will kill enzymes," Smith advised. "Baking, frying, boiling or microwave ovens will also destroy enzyme activity." While he advocated dry mix powders and dietary supplements as the delivery forms with the greatest nutritional return, others have pointed to new technologies, such as specialized coatings and microencapsulation, for possible inroads to processed or functional foods.

Other Technical Considerations

Along with finding the right health focus (digestion vs. systemic benefit) and delivery form (tablet vs. nutrition bar vs. beverage), formulators have other technical challenges and concerns to consider.

Choosing between enzymes derived from animals, plants, fungi or microbes involves several considerations. "For each manufacturer, there may be a specific market of consumers that they are attracting (i.e. vegetarians, pharmacists, veterinarians, dietitians, etc.), and this would initially affect their raw material decisions," Wyant noted, adding one of the first questions American Labs asks its customers is about the enzyme's intended application, because the differentiating factor for enzymes is their effectiveness. "Protease enzymes, for instance, can be sourced from plant, animal, fungal and bacterial sources, each with particular pH and temperature ranges in which the enzyme is active," he explained. "Determining the intended applications for the enzymes and matching them with the individual enzyme characteristics helps us offer the best product to our customers."

Important to any natural health formulation, dosage is an extremely crucial consideration when it comes to enzyme formulation. Unlike other natural product ingredients, enzymes are measured based on activity level, not weight, volume or raw quantity. The activity measure is of an amount of a specific enzyme needed to catalyze a specific substrate over a specific amount of time, and even at a specific temperature and pH level. Given different concentrations of the same enzyme ingredient, the manufacturer/formulator will have to determine which one matches the activity level required for the functional endpoint of the product to be made and which one represents the better price value relative to the different concentrations. For example, simply putting a certain amount of mg of an enzyme in a formula is no guarantee that the activity level of the ingredient is appropriate or consistent to produce the desired effect.

Complicating matters further, there are many different units of measurement at play in the greater enzyme ingredient marketplace. Tina Morris, Ph.D., director of biologics & biotechnology at U.S. Pharmacopeia (USP), said there is a big focus on units of measurement used for enzymes. "There are different units floating around; our goal is to come up with scientifically based units and, wherever possible, have a reference material that aids in the measurements," she said, noting USP is hosting scientific workshop this summer, which will be collaboration between dietary supplement, food and biologics departments at USP, as well as many members of the enzyme industries. "We have many questions about relative assays vs. absolute assays, where International units are available, if there are existing assays that have been used for long time, how units are assigned, and if any assays should be revisited."

Of great use to industry is the Food Chemicals Codex (FCC) which is becoming somewhat of an industry standard, with FCC units making inroads to the greater enzyme market. "FCC has quite a complete monograph on enzyme preparation, which includes most of the enzymes I've seen used in dietary supplements and food products," said Kristie Bowman, M.S., senior scientific associate for USP, which oversees the FCC program. She said the compendium includes info on how enzymes are sourced—animal, plant, fermentation, etc.—as well as info about producing enzyme preparations and adhering to Good manufacturing practices to limit microbial growth and control other production aspects. "Most importantly, FCC has an appendix that is very complete for dietary supplements and foods, of enzyme assays that are commonly used. I don't know of many compendia that have this much info on enzyme and such complete assays, most which have been used for many years in industry, and some which are new—we are always updating them."

The problem with buying enzymes in different units of measurement—papain is sold in many different units of measurement, depending on the company—is you would have to conduct a different assay on each unit-lot in order to know what is being purchased and to compare offers/prices from different companies. "The challenges arise because a lot of vendors choose to use their own units instead of compendium units; this is a marketing thing, which is understandable, but when it comes to direct comparison of enzymes, it becomes difficult for manufacturers to do without a compendium unit assignment," Bowman noted. "This is why we are working hard to assign units that not only work with product, but also with industry."

Likewise, it's important to stick to activity units as the driver of purchase price and not just a guarantee of a certain activity level of a lot sold by weight. Otherwise, the specific gravity could change, which would affect the density and weight, making the price go up, while the activity remains the same.

This seems like a complex trading atmosphere, but Wyant claimed top-notch formulators have become accustomed to this market. "Formulators spend every day working with varying quantities, activities, density specifications, particle sizes, etc. for each raw material they are sourcing," he assured. "Knowledge of how to use these variables and create a product that is functional and practical is well within their grasp."

Even when the right supplier, enzyme materials and price come together, a formulator must decide whether to blend enzymes in-house or buy them already blended.

Smith noted while the demand for single-enzyme products continues, there is a much greater emphasis on enzyme blends in today’s market. Many suppliers offer proprietary blends or help customers develop their own unique blend. "From a marketing standpoint, it is generally better to have a unique, branded enzyme blend than a generic," Smith said, noting there are various issues involved in this decision-making process, including cost, dosage requirements, target audience and competition.

Finally, for those handling the enzyme raw material supply during testing, inspection and production, there are safety concerns. As proteins, enzymes are potentially dangerous if breathed in, which can trigger allergies and other problems. As enzymes are often sold in dry form, it is possible that some material gets airborne. The Enzyme Technical Association (EnzymeTechnicalAssoc.org) offers educational material on the safe handling and workplace procedures for enzymes. For manufacturers new to working with enzymes, an experienced contract packer can be a great help, and some suppliers even offer contract-packing services that could take some of the handling concerns out of the manufacturer's hands. Enzyme Development Corp. noted enzymes are available either in a liquid form or specially processed to minimize the dust.

Potential Applications

Considering there are around 3,000 enzymes known to scientists, Anders Østergård, director of feed, food and specialties for Novozymes, noted only between 50 and 100 enzymes classes are actually commercialized, which includes industrial, chemical, pharmaceutical, nutraceutical and food processing uses. "We are really just scratching the surface of the enzymes found in nature."

In fact, the use of enzymes for digestive or systemic health benefits of consumers is a very small part of the global commercialization of enzymes. The Novozymes catalog of enzymes and their applications reflect this overall market. The enzymes used in food and beverage application largely provide a benefit to the production or process of foods and beverages. This may be in enhancing protein-based flavors or improving the quality or functionality of the end product. For instance, Østergård explained how a phospholipase enzyme can help make two percent more mozzarella or pizza cheeses from a given amount of milk. This shows the potential economic benefit of such enzyme applications, as well as a sustainable benefit due to the need for less milk.

While the enzymes used in such food processing can improve the quality and economics of the final product, the enzymes are not present in the final product for ingestion, thus they have no direct health benefit for the consumer. However, in some cases, enzymes can help extend the freshness of bread or reduce the level of acrylamide—a known carcinogen—in fried or baked foods. These would good indirect enzyme benefits realized by the end consumer.

As production or processing aids, enzymes are useful in a broad range of applications, from foods (dairy, cereal products, meat, fish) and beverages (brewing, juice, wine) to flavorings, sauces, oils/fats and even infant formula. One application in the nutraceutical industry is in producing more palatable and workable soy protein isolate for dietary supplements and functional foods.

Novozymes partnered with Solae to find an enzyme that would help produce a dissolvable, neutral-flavored soy protein ingredient. Improving this area of soy protein characteristics means the ingredient can now be offered as dry powder for use as a ready-to-mix drink mix or in nutrition or energy bars without the production challenges and off-flavor commonly associated with soy protein isolate in the past.

Personal care is even a potential area of application, as Østergård noted enzymes can help soften skin or improve the quality and functionality of eye wash or contact lens solutions, for example.

Smith said the nutraceutical industry in North America continues to be a major growth area for Specialty Enzymes, with other growth areas being bio-ethanol, distilled spirits, brewing, fruit juice, animal feed and waste management.

Due to the range of different enzymes available, the various purposes and applications for these enzymes and the many stability issues present in enzyme preparation and delivery, formulating with enzymes can be different from working with most other natural products, with the exception of maybe probiotics. However, given the many digestive and systemic health benefits, and the countless food and beverage processing benefits, enzymes remain among the most popular natural products ingredients. With all the resources available to help guide quality enzyme formulation and production, these many challenges can be met and benefits realized.