After several entries on additives, today we will discuss enzymes, both those naturally found in dough and those that can be added. Since there are many enzymes, we will divide this entry into two parts. In this first entry, we will cover general aspects and some of the most commonly used enzymes in baking, particularly the essential ones for the process, amylases.

Enzymes: General Aspects

Enzymes are proteins capable of catalysing (facilitating) specific reactions. The most well-known enzymes act by breaking down various chemical components, such as starch (amylases), proteins (proteases), or lipids (lipases). However, there are also enzymes that can bind proteins or starch or facilitate other reactions. Enzymes are present in natural products, and indeed, grains and flours contain a significant amount of enzymes. These enzymes can be either beneficial or detrimental to various processes, and these processes need to be adapted to harness their full potential. Enzyme content can also be enhanced through certain techniques, such as germination. In some cases, commercial enzymes can be added, either because the natural ingredients lack sufficient positive enzymes or because there are beneficial enzymes not naturally occurring in the formulation components.

These products have been known for many years, but their industrial production and competitive pricing did not occur until the late 20th century. Once industrial production was perfected, the world of cereals, particularly in baking, became one of the primary consumers of enzymes within the food industry. Initially, enzymes were extracted from natural substances, such as the stomachs of certain animals or plant products. However, technology now allows for enzyme production from microorganisms, a far more cost-effective method that produces more consistent and homogeneous enzymes with a lower environmental impact. Nevertheless, the first enzymes produced using this technique often had contamination issues with other enzymes. For example, when amylases were obtained, they could be contaminated with proteases. This was a significant problem because in pursuit of a positive effect, there was a risk of encountering a negative one. Enzyme purification techniques have made it possible to obtain highly pure enzymes that do not have adverse side effects at a very competitive price. For scientific studies, even more purified enzymes are available, but at a much higher cost.

The initial applications of commercial enzymes in baking focused on complementing the natural enzymatic activities of flours, resulting in products with more consistent performance. In a later phase, enzymes that were not naturally present in dough ingredients but offered unique advantages were introduced. Currently, one of the main uses of enzymes is as substitutes for certain additives, leading to clean-label products or products free of additives. This is possible because enzymes do not need to be declared on the label since they become inactivated during baking as a result of protein denaturation caused by heat.

There are several factors to consider when using enzymes that may not be as crucial in other chemical reactions.

• Each enzyme has an optimal temperature for its activity and a range of operating temperatures. This means that if an enzyme has an optimum at 40°C, it will also function at 30°C, 50°C, or even 20°C, but as you move away from the optimum, enzymatic activity decreases. If the temperature decreases, you observe a gradual decline, but if it increases, the decline becomes abrupt because enzymes become inactivated at excessive temperatures, as discussed below. Most commercial enzymes have their optima between 40 and 60°C. In the baking process, this factor is crucial because, for example, if we use an enzyme with an optimum at 40°C and want it to work during fermentation, its activity will be much lower if fermentation occurs at reduced temperatures, although, as we will see, time is also an important factor.
• Each enzyme has a temperature of inactivation, beyond which it irreversibly loses its activity. In general, we want enzymes to be active during the baking process but not on the final product. For instance, if amylases are not inactivated during baking, they would degrade the starch in the crumb, leading to a loss of the bread’s internal structure and even liquefaction. Therefore, we must select enzymes that become inactive during the baking process. In this case, it should be noted that although the oven is maintained at temperatures close to 200°C, the inside of the dough does not exceed 100°C.
• Each enzyme has an optimal pH for its activity. Like temperature, moving away from this pH will progressively reduce enzymatic activity. Most commercial enzymes have optima close to neutrality (pH 7). It should be noted that dough usually has a slightly acidic pH (around 6), but the use of sourdough can reduce this pH to values near 4.5, so most enzymes do not perform equally in both conditions. On the other hand, enzymes that may be of great interest for breadmaking may not work in products like cakes due to the higher pH of cake batters.
• Enzymes do not get depleted as certain chemical reagents do. This means they will continue to act as long as there is substrate available. While it is true that when the substrate level decreases significantly, enzymatic activity slows down, in baking processes, this hardly occurs. The consequence of this is that the greater the processing time, the greater the enzymatic activity. Therefore, if we change kneading, resting, or fermentation times in doughs with high enzymatic activity, the effects can be more pronounced than in doughs with lower enzymatic activity due to changes in their enzymatic actions.
• Enzymes have specific actions, meaning that there are different types of amylases, proteases, lipases, and each type has a distinct action that will influence doughs differently. This point will be examined in detail in the analysis of each enzyme.

Amylases

Naturally Present in Flour

Amylases are enzymes capable of breaking down starch, and they are naturally present in grains. Additionally, these enzymes are crucial in the breadmaking process, as they are responsible for generating fermentable sugars from starch granules. These sugars serve as food for yeast, facilitating fermentation and producing carbon dioxide, which is essential for leavening dough and increasing bread volume. Therefore, if there are insufficient amylases, it may be necessary to add external amylases. Conversely, an excess of amylases can also be detrimental as it would excessively hydrolyse the starch, resulting in less consistent crumb texture and darker bread due to excess sugars.

In grains, there are two types of amylases, β-amylases and α-amylases. β-amylases are produced during grain maturation, and there are usually no issues with a lack of these enzymes because the amount present in flours is generally sufficient for their intended role. β-amylases work by cutting starch every two glucose molecules, generating maltose (two glucose units connected by an α-1-4 bond). These enzymes stop working when they encounter an α-1-6 bond (a branching point in starch chains). Maltose is a fermentable sugar that yeast can utilize, so one might think that the presence of β-amylases alone would be sufficient. However, the ends of starch chains are very short, and after β-amylase activity, a significant portion of the starch remains unhydrolyzed, with the amount of maltose generated being insufficient.

α-Amylases are produced in the later stages of grain maturation, and grains that have not matured adequately tend to have an insufficient quantity of these enzymes. If the grain starts to germinate, amylase production increases, which can also be problematic. This enzyme also breaks α-1-4 bonds, like β-amylases, but does so in a disorderly manner, skipping α-1-6 bonds and generating smaller starch fragments called dextrins. The initial effect of α-amylases is to reduce the viscosity of pastes or doughs made with starch because larger starch sizes have more thickening power. Another important effect is creating new ends where β-amylases can begin to act. Therefore, a combined effect of both enzymes allows for the generation of an adequate amount of sugars for fermentation (mostly maltose) without excessively hydrolysing starch.

In general, flours have sufficient β-amylases but may have a somewhat insufficient quantity of α-amylases, so it is usually advisable to incorporate external α-amylases. To assess the content of α-amylases, the falling number test is often used, which has been discussed in a previous entry.

Regarding amylase activity in flours and doughs, another factor to consider is the presence of damaged starch. Intact starch granules cannot be attacked by the amylases present in the flour, or they do so too slowly to be noticeable in conventional breadmaking. However, during the milling process, some of the starch can become damaged, allowing amylase action on it. Fortunately, damaged starch is relatively consistent (between 3% and 8%), with slightly higher levels in harder wheat varieties compared to softer ones. This level of damaged starch is adequate for the generation of the necessary maltose for fermentation, but not excessive. If the amount of damaged starch were higher, the action of amylases would lead to less consistent and stickier doughs, as well as darker breads, as the generated sugars would participate in reactions that create the brown colour of crusts.

Added Amylases

As mentioned, it is common for the amylase activity of flours to be insufficient for proper breadmaking. To compensate for this deficiency, external α-amylases are often used. These enzymes can be added directly in flour mills, but it is also very common for bread improvers to incorporate these enzymes. When selecting these amylases, the optimal temperature and pH for their activity must be taken into account. There are both fungal and bacterial amylases. Bacterial amylases have an optimal pH slightly further from that of doughs than fungal amylases, so the latter are generally preferable. However, the significant issue with bacterial amylases is that they have a higher optimal temperature and a higher inactivation temperature. This results in their functionality in dough being more limited compared to fungal amylases. Additionally, some bacterial amylases can survive the baking process without becoming inactivated, leading to bread degradation. For this reason, bacterial amylases should generally be excluded, and fungal amylases should be chosen to complement the amylase activity in flours. These fungal amylases are usually denatured at 60°C, thus becoming inactivated before starch gelatinizes during baking. However, as we will see later, there are special bacterial amylases that can have a positive effect on bread quality.

Some bacterial amylases have a slightly lower inactivation temperature than usual and are fully inactivated during baking. These are known as intermediate stability amylases. While they may not be ideal for complementing the amylase activity of flours to generate sugars for the fermentation process due to their slightly higher optimal temperature, they can partially hydrolyse starch when it has gelatinized during baking. Thanks to their higher thermal resistance, they can act at higher temperatures than starch gelatinization temperatures. Once starch has gelatinized, it becomes fully accessible to enzymatic action, as this process breaks down the granular structure. The action of these amylases on starch is brief because they become inactivated as the temperature continues to rise. However, this action is sufficient to reduce retrogradation phenomena and slow down bread staling (hardening). Thus, the use of these enzymes could reduce the need for monoglycerides, the most commonly used additive for this purpose.

If commercial enzymes are not desired, another option to compensate for low amylase activity is the addition of flours made from sprouted grains or malt flours. The germination process triggers enzyme production, particularly amylases. Therefore, flours from germinated grains will be rich in these enzymes. However, care must be taken regarding two aspects. Firstly, the germination conditions, as if the process is too short, enzymatic activity may be reduced, and secondly, the drying temperature. For the germination process, grains need to be moistened. This excess moisture must be removed before milling, which requires subjecting the grains to a drying process. If this process occurs at a high temperature, many of the produced enzymes will become inactivated. To achieve flours with high enzymatic activity, a high degree of germination should be reached, and the grains should be dried at mild temperatures before milling. Amylases present in grains are inactivated at 70°C, so they can slightly hydrolyse the starch gelatinized during baking. This means that an excess of these enzymes could be more problematic than an excess of fungal amylases.

Other Enzymes

There are other enzymes capable of acting on starch. Some of these are widely used in the starch industry to obtain maltodextrins or glucose syrups. However, in general, no utility for these enzymes in baking processes has been found, except for a few exceptions. For example, there are enzymes that can hydrolyse α-1-6 bonds (branching points in amylopectin chains), known as pullulanases. It should be noted that α and β-amylases can only hydrolyse α-1-4 bonds, so pullulanases are necessary for complete starch degradation. There are also enzymes that have the opposite effect, i.e., they can create α-1-6 bonds and, therefore, branches. These are known as branching enzymes. The commercial amylase enzyme that has found some application in baking, in addition to α-amylases, is glucoamylase. This enzyme acts in a manner very similar to β-amylases, but instead of cutting every two glucose units, it cuts every single one. Thus, instead of generating maltose, it produces glucose, a sugar that ferments more rapidly than maltose by yeast. Glucose has a higher sweetening power than maltose, but most of it is consumed by yeast, so the sweetness is barely noticeable in the final product. Additionally, glucose is more effective in Maillard reactions, leading to a more golden colour in bread, a characteristic that is apparent in breads made with doughs containing this enzyme.