Many food product designers consider enzyme use new and innovative. While this is true for many categories, the baking industry actually has a long history of enzyme study and application. In fact, some references to the use of added enzymes in bakery foods are over 100 years old.
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Even without this track record, enzymes are appealing functional ingredients for a variety of reasons. Enzymes are, for example, naturally occurring components of many bakery ingredients. If an enzyme is added, it often is destroyed by the heat of the baking process. In both cases, designers can obtain the functional benefits of the enzyme while maintaining a "clean label" image for the finished product. Enzymes also are specific to a particular function, eliminating concerns about undesired effects.
Nevertheless, getting the most out of enzymes in bakery products requires some planning on the part of the designer and a better understanding of what enzymes can do.
Enzymes are named by adding the suffix "ase" to the end of the substrate. For simplicity, the substrate's name often is abbreviated. In baking applications, the general types of enzymes most commonly used are carbohydrases, proteases and lipoxygenases.
The alpha-amylase enzyme hydrolyzes starch into soluble dextrins. These dextrins may subsequently be hydrolyzed by beta-amylase to yield maltose, and/or amyloglucosidase to yield glucose. Because starch exists as a tightly packed granule, amylases must act upon starch granules that are damaged (as many are during flour milling) or on granules that have been gelatinized by moisture and heat (such as when a dough is mixed and baked).
The sugars resulting from amylase activity act as food for yeast in yeast-raised products. As a result, the presence of these enzymes in the proper proportions is critical to carbon dioxide generation. Most flour naturally contains both alpha- and beta-amylase. The beta-amylase is, however, the only one naturally present in sufficient quantities. Thus, controlling the gassing power of the dough requires added alpha-amylase.
Amylases also can affect the consistency of a dough. Damaged starch granules absorb more water than intact granules. This ability is reduced when the damaged granules are acted upon by amylases. With their ability to immobilize water reduced, the damaged granules release free water which softens the dough and makes it more mobile.
A third function of amylases is their ability to retard staling. Over time, the crumb of baked products firms due to a complex set of changes that includes recrystallization (or retrogradation) of amylopectin in the starch. By hydrolyzing the amylopectin into smaller units, bacterial alpha-amylase can maintain softness and extend shelf life.
One theory behind this suggests that amylopectin still crystallizes at the same rate with added enzymes, but that the shortened chain length maintains greater flexibility and softness when crystallized. Another theory is that the shortened amylopectin chains have a lesser tendency to retrograde. Either way, the enzyme must continue to hydrolyze starch after baking is completed. The fact that bacterial alpha-amylase is more thermally stable than other alpha-amylase sources is the reason it is used.
Because the enzyme is active in the finished baked product, it is possible for the enzyme activity to go too far. Rather than maintaining softness, the crumb can actually become gummy. The starting enzyme dosage is critical to preventing this. For even greater assurance against overdosing, amyloglucosidase or pullulanase may be added along with the alpha-amylase. These enzymes don't contribute to anti-staling when used alone, but help prevent gumminess when combined with the amylase.
A final use for amylases in bakery products is for replacing potassium bromate, an oxidizing agent that strengthens gluten strands. Strengthened gluten produces a dough with improved gas retention and, consequently, higher volume in the finished product.
Based on various health studies, bromate use is on a sharp decline. Other oxidants &#; such as ascorbic acid &#; can promote comparable volume, but they don't provide a direct match for bromate. To compensate, alpha-amylase can be added with ascorbic acid to improve the volume and increase the quality of the crumb. Bakeries may either add alpha-amylase and ascorbic acid separately or select a custom blend featuring an optimized mixture of the two components.
Amylases are not the only carbohydrases useful in bakery products. Pentosanases also can be added to improve quality. Both wheat and rye flour contain pentosans. These non-starch polysaccharides are highly hydrophilic and contribute significantly to the water absorption properties of a dough. In wheat flour-based products, pentosans also interfere with volume development.
Adding pentosanase to a wheat flour-based product can improve product volume by hydrolyzing the pentosans present. At the same time, though, hydrolyzed pentosan will release water, making the dough very slack. When using pentosanase, the water absorption of the dough must be adjusted to compensate. If the dough is too slack, not only will it be difficult to machine, but the volume-building benefits of the pentosanase will not occur.
In rye bread, the pentosans in the rye flour are critical to building structure since rye flour's gluten content isn't sufficient. If pentosan content is too high, though, it will compete for water with the starch and prevent it from swelling and gelatinizing properly. Pentosanase will help control the pentosan content so there is enough to build structure, but not so much as to interfere with the starch functionality.
Pentosanases that hydrolyze cellulose also are available. These may be added to high-fiber bakery products to help improve their eating qualities by breaking up the long cellulose chains that contribute to gritty mouthfeel.
Nevertheless, protease could be added to an entire dough later, at the mixing stage. This won't reduce the mixing time because the enzyme will not have had enough time to hydrolyze much gluten. Still, as hydrolysis occurs through shaping, floor time and proofing, the protease will help improve the flow of the dough. This procedure might be used to eliminate short pan fills in a straight (non-sponge) dough system or to help the pan flow of buns and English muffins.
Another application for proteases is in replacing sodium sulfites in cracker doughs. Cracker doughs contain low levels of fat and water, making them rather stiff. This stiffness makes it difficult to laminate the dough into layers and to sheet it to cracker thinness. Sodium sulfites hydrolyze the disulfide bridges on the gluten molecule, reducing its resistance to extension and making the resulting dough more plastic.
Sulfites have undesirable side effects, however. They break down vitamin B2, inhibit browning reactions that are desirable in baked products, and are a marketing no-no because some consumers exhibit allergic reactions to the substance. In fact, many countries have banned or are considering banning sulfite's use in bakery products. Adding a protease to the formula and allowing sufficient time for the enzyme to act (sulfites, by comparison, react more rapidly) can achieve the desired workability in the dough without the negative side effects.
In a way, lipoxygenases offer results similar to those obtained with dough strengtheners such as sodium stearoyl-2-lactylate, but they also offer additional benefits. Although the exact mechanism behind it is not fully understood, lipoxygenase can bleach fat-soluble flour pigments to produce a whiter crumb in finished bread and rolls.
Understanding what different types of enzymes do to bakery products is the first step in enzyme selection. Considering how specific enzyme action is, once the desired results are determined, the enzyme to use will be a straightforward decision. Other factors in enzyme selection and use aren't so easy. These include the enzyme source and form, the strength of the enzyme activity and how much to use, and the conditions under which the enzyme will be used and handled.
Amylases used in bakery foods come from three primary sources.
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 issue of Food Product Design.)
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.
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.
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.
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.
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.
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.
Serine proteases often are called alkaline proteases because their activity is optimum above pH 7.5.
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.
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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.
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.
The use of enzymes dates from much longer than their ability to catalyse reactions was recognised and their chemical nature was known. The first completely enzymatic industrial process was developed in the years . Starch processing, which is undertaken in two steps, involves liquefaction of the polysaccharide using bacterial α-amylase, followed by saccharification catalysed by fungal glucoamylase.
Developments in process technology allied to the use of recombinant techniques during the last decades allowed for considerably improved yields by fermentation increased stability and altered specificity and selectivity of enzymes. Those techniques thrust forward and are continuing to broaden the applications of enzymes in food technology and many different areas.
There are two scenarios regarding the use of enzymes, either the enzymes are used to convert the raw material into the main product, or the enzymes are used as additives to alter a functional characteristic of the product. In the first case, the enzymatic process is undertaken in optimised and controlled conditions to enhance the catalytic potential of the enzyme, whereas in the second situation it is more difficult to assure optimal conditions and to control the enzymatic reaction.
The development of the bread process was an important event in mankind. An important aspect that contributed to the evolution of the baking market was the introduction of industrial enzymes in the baking process, where bakery enzymes represent a relevant segment of the industry.
Baking is a common name for the production of baked goods, such as bread, cake, pastries, biscuits, crackers, cookies, pies and tortillas, where wheat flour is both the most essential ingredient and a key source of enzyme substrates for the product. Even though based on cereals other than wheat, baked goods such as gluten-free products or rye bread are also considered to be baked products.
Bread is usually made from wheat flour as raw material, which is a mixture of starch, gluten, lipids, non-starch polysaccharides, and enzymes. After flour, yeast, and water are mixed, complex biochemical and biophysical processes begin, catalysed by the wheat enzymes and by the yeast, characterising the dough phase. These processes go on in the baking phase, giving rise to bread.
Extra enzymes added to the dough improve control of the baking process, allowing the use of different baking processes, reducing process time, slowing-down staling, compensating for flour variability and substituting chemical additives.
Starch is the main component of products such as bread and other bakery goods and is added to different foods, acting as a thickener, water binder, emulsion stabiliser, gelling agent and fat substitute. It is the most abundant constituent and most important reserve polysaccharide of many plants, including cereals, occurring as intracellular, semi-crystalline granules. On a molecular level, its major components are the glucose polymers amylose and amylopectin.
Amylose is an essentially linear molecule, consisting of up to glucose units with α-(1,4)-glycosidic bonds. On the other hand, amylopectin is a highly branched polysaccharide constituted of short α-1,4 linked linear chains of 10&#;60 glucose units and α-1,6 linked side chains with 15&#;45 glucose units, containing on average 2 million glucose units.
Baking comprises the use of enzymes from three sources: the endogenous enzymes in flour, enzymes associated with the metabolic activity of the dominant microorganisms and exogenous enzymes which are added in the dough. The supplementation of flour and dough with enzyme improvers is a usual practice for flour standardisation and also as baking aids.
Enzymes are usually added to modify dough rheology, gas retention and crumb softness in bread manufacture, to modify dough rheology in the manufacture of pastry and biscuits, to change product softness in cake making and to reduce acrylamide formation in bakery products. The enzymes can be added individually or in complex mixtures, which may act in a synergistic way in the production of baked goods [60-62], and their levels are usually very low.
Enzymes as technological aids are usually added to flour, during the mixing step of the bread-making process. The enzymes most frequently used in bread-making are the α-amylases from different origins. Amylases and other starch-converting enzymes The industrial processing of starch is usually started by α-amylases (α-1,4-glucanohydro&#; lase). Most of the starch-converting enzymes belong to the α-amylase family or family 13 glycosyl hydrolases (GH), based on amino acid se α-Amylases are endo-enzymes that catalyse the cleavage of α-1,4-glycosidic bonds in the inner part of the amylose or amylopectin chain. The end products of α-amylase action are oligosaccharides, with an α-configuration and varying lengths, and α-limit dextrins, which are branched oligosaccharides. These enzymes can be obtained from cereal, fungal, bacterial and biotechnologically altered bacterial sources.
Differences in the number of binding sites and location of catalytic regions determine the substrate specificity of α-amylases, the length of the oligosaccharide fragments released after hydrolysis and, consequently, the carbohydrate profile of the final product. The increased levels of reducing sugars lead to the formation of Maillard reaction products, intensifying bread flavour and crust colour. In addition, these enzymes can improve the gas-retention properties of fermented dough and reduce dough viscosity during starch gelatinisation, with consequent improvements in product volume and softness. Certain amylases are able to decrease the firming rate of bread crumb, acting as anti-staling agents.
Amylase-containing anti-staling products typically consist of bacterial or fungal α296 Food Industry amylases with intermediate thermostability. In this context, one of the most effective anti-staling amylases is the Bacillus stearothermophilus maltogenic α-amylase. The anti-staling action of amylases has been attributed to the modified retrogradation behavior of the hydro&#; lysed starch. Yet, other researchers describe the effect of the interference of the low molecular weight dextrins with starch-starch and/or gluten-starch interactions.
Proteases can be subdivided into two major groups according to their site of action: exopeptidases and endopeptidases. Exopeptidases cleave the peptide bond proximal to the amino or carboxy termini of the substrate, whereas endopeptidases cleave peptide bonds distant from the termini of the substrate. Most of the proteolytic activity of wheat and rye flours corresponds to aspartic proteases and carboxypeptidases, which are both active in acid pH. Additionally, aspartic proteases of wheat are partly associated with gluten. Nevertheless, the proteolytic activity of sound, ungerminated grain is normally low.
Proteases are used on a large commercial scale in the production of bread, baked goods, crackers, and waffles. These enzymes can be added to reduce mixing time, to decrease dough consistency, to assure dough uniformity, to regulate gluten strength in bread, to control bread texture and to improve flavor. In addition, proteases have largely replaced bi-sulfite, which was previously used to control consistency through reduction of gluten protein disulfide bonds, while proteolysis breaks down peptide bonds. In both cases, the final effect is a similar weakening of the gluten network. In bread production, a fungal acid protease is used to modify mixtures containing high gluten content. When proteases are mixed in the blend, it undergoes partial hydrolysis becoming soft and easy to pull and knead. Proteases are also frequently added to dough preparations. These enzymes have a great impact on dough rheology and the quality of bread possibly due to effects on the gluten network or on gliadin.
Proteases are also applied in the manufacture of pastries, biscuits, and cookies. They act on the proteins of wheat flour, reducing gluten elasticity and therefore reducing shrinkage of dough or paste after molding and sheeting; for instance, hydrolysis of glutenin proteins, which are responsible for the elasticity of dough, has considerable improving effects on the spread ratio of cookies.
Hemicellulases are a diverse class of enzymes that hydrolyse hemicelluloses, a group of pol&#; ysaccharides comprising xylan, xylobiose, arabinoxylan, and arabinogalactan. This group includes xylanase or endo-1,4-β-xylanase (4-β-D-xylan xylanohydrolase), a glycosidase that catalyses the endohydrolysis of 1,4-β-D-xylosidic linkages in xylan and ara&#; binoxylan. Xylanase, also designated endoxylanase, was originally termed pentosanase. A wide variety of xylanases have been reported from a plethora of microorganisms including bacteria, archaea, and fungi. These enzymes are mainly classified in the glycosyl hydrolase (GH) families 10 and 11, although putative xylanase activities have been reported in GH families 5, 7, 8 and 43. GH10 xylanases are regarded to have broader substrate specificity and release shorter fragments compared to GH11 xylanases, while the latter enzymes are more susceptible to steric hindrance by arabinose substituents.
Xylanases were introduced to the baking segment in the years and are most often used combined with amylases, lipases, and many oxidoreductases to attain specific effects on the rheological properties of dough and organoleptic properties of bread. These enzymes have also been used to improve the quality of biscuits, cakes and other baked products. The most favorable xylanases for bread-making are those that preferentially act on WU-AX and are poorly active on WE-AX, because they remove the insoluble arabinoxylans which interfere with the formation of the gluten network, giving rise to high molecular weight solubilised arabinoxylans, resulting in increased viscosity and thus enhancing dough stability.
As a consequence, a more stable, flexible and easy to handle dough is obtained, resulting in improved oven spring, larger loaf volume, as well as a softer crumb with improved structure. Moreover, the addition of xylanases during dough processing is expected to increase the concentration of arabinoxylo-oligosaccharides in bread, which have beneficial effects on human health.
Although both enzymes had a positive effect on loaf volume, psychrophilic GH8 xylanase was apparently much more efficient than the mesophilic enzyme from the same family, because much lower concentrations of the former enzyme were required to produce a similar increase in bread volume.
Lipases or triacylglycerol acylhydrolases hydrolyse triacylglycerols (TAG) producing monoacylglycerols (MAG), diacylglycerols (DAG), glycerol and free fatty acids. These enzymes are widely found in nature. Besides TAG lipases there are phospholipases A1, A2, C, D, and galactolipases. Even though they are present in all cereal grains; lipase activity of white flour is usually low enough to avoid rancidity due to hydrolysis of native lipids and of baking fat. The use of lipases in the baking segment is much more recent in comparison to α-amylases and proteases.
The first generation of commercial lipase preparations was introduced to the market in the years and recently a third generation became available. The latter are protein engineered enzymes, claimed to give a better effect in high-speed mixing and no time dough processes. Moreover, third generation lipases have a lower affinity for short-chain fatty acids, which reduces the risk for off-flavour formation on account of prolonged storage of the baked goods and the use of butter or milk fat in baked products. Lipases (TAG lipases) of the first generation are 1,3-specific, removing preferentially fatty acids from positions 1 and 3 in TAG. These enzymes can improve dough rheology, increase dough strength and stability, thus improving dough machinability. In addition, lipases lead to an increase in volume which results in an improved, more uniform crumb structure; hence a softer crumb is obtained.
The second-generation lipases act simultaneously on TAG, diacylgalactolipids and phospholipids, producing more polar lipids, providing a greater increase in volume, better stability to mechanical stress on the dough, and a fine, uniform bread crumb structure compared to the first generation lipases. Moreover, a third-generation lipase was found to increase expansion of the gluten network, increase the wall thickness and reduce cell density, enhancing volume and crumb structure of high fibre white bread.
In this context, the roles of lipids and surfactants in bread-making have been extensively reviewed elsewhere. The addition of lipases has been claimed to retard the rate of staling in baked products. The effect of these enzymes has been attributed to in situ production of MAG following TAG hydrolysis, although this mechanism is not completely accepted because the amount of MAG would be insufficient to account for the antistaling effect.
Lipases may also be used for the development of particular flavors in bakery products. The effect of a third generation lipase on the quality of high-fiber enriched brewer&#;s spent grain bread has been evaluated. The enzyme produced beneficial effects during bread making, positively affecting loaf volume, staling rate, and crumb structure.
The application of lipase and MAG to produce fibre enriched pan bread using the straight dough method was assessed. The use of lipase dosages up to 50 ppm and MAG up to 2% indicated the possibility of replacement of MAG by lipases in fibre enriched pan bread. Recently, the effects of two lipases and DATEM on the rheological and thermal properties of white and whole wheat flour doughs were compared.
Lipases were able to cause modifications in the dough components (gluten proteins and starch). The enzymes improved dough handling properties to a similar or greater extent than DATEM, increasing dough stability, maximum resistance to extension and hardness, and decreasing softening degree and stickiness. The possible role of lipases in delaying starch retrogradation was indicated by the greater extent of formation of amylose-lipid complexes promoted by lipases in comparison to DATEM.
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