Understanding Enzyme Function in Bakery Foods

Nature of the ingredient

Starch segregation

Harnessing protein pruning

Bond-building catalyst

Application intricacies

1. Malt ingredients. As previously mentioned, flour contains naturally occurring amylases. The same is true for cereals other than wheat. When a cereal kernel becomes moist and germinates, it experiences a dramatic increase in alpha-amylase. Consequently, malting grains such as barley and wheat can serve as the basis for many alpha-amylase-containing ingredients. (For a discussion of the malting process, see "Grains: The Bottom of the Pyramid at the Center of Attention," in the September 1994 issue of Food Product Design.)
2. Malt flour is most frequently used by millers to standardize the alpha-amylase content of wheat flour, although it is also often found as an ingredient in crackers and certain breads. It is made from wheat or barley that has been germinated, dried and ground to flour fineness.

3. Malt extracts and syrups start with germinated barley. Rather than grinding the kernels after drying, these ingredients are made through a series of liquid extraction and concentration steps that preserve the grain's alpha-amylase activity.   Diastatic malt syrups are made the same way, but start with a blend of corn and barley. This causes diastatic syrups to have less of the malt flavor contributed by regular syrups and extracts, yet provide the same level of enzyme activity.
     The non-diastatic malt syrup process is similar, but produces an ingredient without the amylase activity. This is then used for non-enzyme related benefits such as flavor and improved crust color.

4. Fungal amylase. During growth, certain fungi synthesize alpha-amylase. Cultures of Aspergillus oryzae are extracted, concentrated and dried to yield fungal amylases. These are available both in ready-to-use tablet form and blended to a predetermined activity with flour or starch to yield a powdered form. Fungal amylases can be used to standardize wheat flour, but are most often added at the production facility to aid with dough conditioning.

5. Bacterial amylase. Certain bacteria, such as Bacillus subtillis, also synthesize alpha-amylase. This can be extracted and dried much like fungal amylases. Bacterial amylases, however, tend to be more thermally stable and are, therefore, useful for maintaining softness in finished baked products.
     Like amylases, proteases for bakery applications can be extracted from both fungi and bacteria -- most often with the same species used for alpha-amylase production. Different types of protease have different catalytic mechanisms. The different mechanisms primarily control how the enzyme responds to different pH conditions.
6. Acid proteases can be found in flour and have a low pH optimum. They are thought to mellow gluten during long-term, low-pH fermentation of saltine cracker sponges.

7. Sulfhydryl proteases are found in many grain-based ingredients such as flour and malt. They also are extracted from pineapple stems (bromelain) and papayas (papain). Sulfhydryl proteases have a pH optimum range from around 3.5 to nearly 8.5.

8. Serine proteases often are called alkaline proteases because their activity is optimum above pH 7.5.

9. Neutral proteases make up most of the commercially available proteases. Here, the pH is optimum in a narrow range around 7.   Lipoxygenases aren't available in concentrated forms like proteases and amylases. They are added as a natural constituent of full-fat and defatted soy flour. These flours often are offered with other functional ingredients such as calcium peroxide for additional oxidation, dicalcium phosphate for dough conditioning, and corn flour to improve absorption and mix tolerance.

   Activity problems

     Because they are a naturally occurring component of soy flour, lipoxygenase activity is not as standardized as it is with the tablets, powders, etc. available with amylases and proteases. Though these forms sport standardized activity levels, product designers still may be confused by the different methods of activity measurement. The amylose activity of malt extracts and syrups, for example, is typically expressed as degrees Lintner, while concentrated amylase sources are often expressed in Sandstedt-Kneen-Blish (SKB) units.
     On top of the tremendous number of standardized tests, individual enzyme suppliers often have a custom method of determining enzyme activity. This presents a challenge to product designers trying to compare activities in order to predict usage levels and cost impact.
     The goal of most methods of measuring enzyme activity is to determine how quickly the enzymes convert substrate molecules to product molecules. Because of this, the activity measurements often have little to do with the enzyme's activity in actual use, particularly in baked products. Designers will probably wish to create their own assay by testing enzymes at different levels in actual doughs. The observed effects can then be related to the amount of enzyme added.
     By using the level of activity per gram of enzyme as the measuring unit, product designers will have a common basis for comparing enzymes. In addition, the activity measurement will include a weight that can be directly related to the price of the ingredient in order to determine the cost of a given degree of effectiveness.

   A handle on handling

     When creating a test for comparing enzyme activity and when preparing to put enzymes to work in the formula, remember that conditions the dough encounters through the process will greatly affect enzyme activity.
   -- Time is critical for successful application of many enzymes. Put simply, the chemical reaction must have enough time to proceed. An enzyme's catalytic reaction can, of course, be sped up by increasing the enzyme level to increase the amount of available catalyst. However, this can be expensive and, in the case of bacterial amylases for shelf life extension, be impossible due to detrimental effects in the finished product.
     Keep in mind also that amylases can only act on damaged or gelatinized starch granules. A certain amount of mixing and/or dough development will be required before these enzymes begin to work. A protease will start to act as soon as a dough is wetted.
   -- Temperature influences enzyme activity in both a positive and negative way. Every 18 degree F increase in dough temperature increases the enzyme activity up to two-fold. On the down side, the same temperature increase also will accelerate the rate of enzyme denaturation by a factor anywhere from 10- to 30-fold. At a high enough temperature, the rate of denaturation catches up with the reaction rate, slows it and eventually stops it. Just as the time and enzyme amount must be optimally balanced, so must the time and temperature. A longer reaction time can actually increase the efficiency of the enzyme conversion at a lower temperature.
   -- Acidity, or pH, affects enzyme activity. Different enzymes, and even enzymes from different sources, have optimum pH ranges under which they are most active. This was previously discussed for different proteases, but also is true for amylases.
     When formulating, designers must not only be aware of the starting pH of the formula, but how it changes over time. For example, as chemical leaveners are consumed, the overall dough pH may be altered out of the optimum range for the enzyme. The same is true for yeast-leavened products, as the pH can change dramatically as fermentation proceeds in products such as crackers and bread.
     Care also is necessary when adjusting the formula pH. Cocoa powder and other chocolate-flavored ingredients require an alkaline system for optimum flavor. Adjusting such a system to be more acidic for the enzyme can adversely affect the flavor. Color development is strongly related to pH, and any alterations will affect a product's crust color.
   -- Salt level can affect the enzyme's activity because salt can help stabilize certain enzymes. The opposite is true, however, for proteases, which are inhibited by high salt concentrations. This could be the result of salt making gluten less available to the action of the enzymes.
     If salt levels can't be adjusted, the order of addition can overcome this limitation. In a sponge-and-dough bread, for example, enzymes can be added to the sponge. Because salt won't be added until the dough stage, the enzymes will have more time to react uninhibited. Salt also can be added to later steps in multiple-stage mixing procedures for other products, but the time between stages isn't nearly as significant as it is between a sponge and a dough.
     Certain enzymes may require ionic co-factors to be active. Many carbohydrases will not function without calcium ions. Zinc is necessary for neutral fungal proteases.
     Enzymes are indeed a highly specific, useful collection of ingredients for bakery products. Enzyme activity itself is useful, and many enzyme applications offer clean-label advantages. Although the number of different enzymes and the cacophony of different activity measurement methods may seem intimidating, product designers can sort through this to determine the best enzyme and the conditions that enzyme requires in the formula and during processing for maximum effectiveness. All it takes is a new understanding of these old ingredients.

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