Digital Re-print March | April 2013

Additives for flour standardisation - Part I: Enzymes

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FEATURE

Part I: Enzymes
by Lutz Popper, Mhlenchemie GmbH & Co. KG, Germany

Additives for flour standardisation -

n biological systems, all the conversion processes can take place quickly at relatively low temperatures and mild chemical conditions, because enzymes help to run the reactions with lower energy input. Because of the ability to perform very complex reactions under these mild conditions, enzymes are produced in industrial scale, mostly by micro-organisms. The development of new enzymes in short succession all around the world is fueled by increasing competition. High quality and low cost products manufactured with the help of these enzymes have the chance to compete better in the market. Enzymes are used in many areas of the food industry. In contrast to most other food application, enzymes used in the flour industry do not show their effects at the moment they are added, that is, right in the mill. In order to see the effect of enzymes in flour, the baker must add water. This problem of time and place is the general challenge in the flour improvement business, but it gets even more complex when it comes to enzymes. But enzymes also have definite advantages as they are specialised on distinct effects, used in very small dosages, natural and completely deactivated under baking conditions. As with all the concentrated natural substances, enzymes pose the risk to cause aller-

gic reactions. Therefore, it is advised that the employees in contact with enzymes should wear gloves, mask and goggles. Although with a lower probability because of the dilution in flour or bread improvers, the same risk is present for bakers. Therefore, enzyme producers are trying to manufacture preparations that emit less dust. For a long time, - and -amylase were thought to be the only enzymes that could be used in the milling industry. This view has changed dramatically since the introduction of hemicellulases two decades ago, and has now received another blow through the success of lipolytic enzymes. There are many more enzymes (Table 1) that still play niche roles for certain applications, but which may turn out one day to be as versatile as the aforementioned types.

most enzymes, amylases also act on substances that are well in contact with water. Alpha-amylase breaks down unbranched moieties of starch molecules, releasing dextrins. These dextrins act as substrate for beta-amylase and glucoamylase, which in turn produce sugars like maltose and glucose that can be directly used by yeast. By the action of amylases, the dough viscosity is decreased (water released from the starch), the fermentation power and the volume are increased, taste and colour are improved, the crumb softness is retained and the shelf life is extended.

Amylases
The most used types of amylases in flour industry are alpha-amylase, beta-amylase and amyloglucosidase (glucoamylase). Like

Figure 1: Enzyme production scheme

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FEATURE FEATURE is sensitive machinery, motors or anything electric and adversely affected by water. The technology, which is the first of its kind in the UK, uses a continuous flow heating coil system to heat water to such a high degree that it becomes extremely hot vapour. The emitted dry steam contains minimal moisture and efficient cleaning capability is produced from the steam pressure made on the surface area. As well as being applicable to a wide range of work settings, steam can be used to undertake innumerable cleaning tasks. The system can be used on feeding, mixing and blending vessels, machinery, conveyor belts, rollers, pipelines and also general floor areas, storage spaces and much more. As it is applied to the surface, dry steam leaves very little residue and can almost touch dry, especially when compared gram of a 50,000 SKB/g amylase per 100 includes the fully auditable dry steam beltkg of flour). There is much native enzyme sanitation unit (BSU) which less cleans conveyor in flour with level, Fallingsaving Numbers 400 belts to allergen up to above 3 million seconds, therefore 3 grams or more of the litres of water per annum. same amylase may has be used. On the other The organisation also developed a hand, steam in flours with very FNproduction values, using central system for low food trace amountsareas. of amylase 0.1-0.2 grams and packaging This (like is much like a of 50,000 SKB/gwhich amylase) will not cleaning affect the central vacuum, facilitates but may have the a beneficial the byFN simply plugging steam effect hoses on into dough properties volume yield. central steam pipesand without the need for handling cleaning machines. The sophisticated equipment can be used for the Glucoamylase cleaning of heavy parts and foror plastic Also called amyloglucosidase previcleaning. The ously parts gamma amylase, thismachines enzyme start breaks from a 3kW single phase unit and starch to its smallest building blocks, glucose. reach on up the to 144kW units avail- of It also can work branching points able in to electric, oil or amylopectin, as opposed alpha-amylase. gas heated coils. With this property, glucoamylase helps improving the browning of baked products and stabilises the fermentation in prolonged fermentation processes. There is no viscosity lowering effect of this enzyme because it leaves the large starch molecules basically unaffected, at least within the relatively short time of baking processes.

Is all steam cleaning the same?

So steam is simply steam? Well actually, no. Steam can be produced in a range of different grades, each matching different industry applications. Picking the cleaning method best suited to your operation depends upon a number of options including the nature of the surface to be cleaned and the type of material or residue found on crushing or milling apparatus, conveyors and elevator pits and silos. Enzyme active malt flour To date,need conventional CIP systems have Seeds enzymes to start with their vital tended to rely on traditional boiler activities, therefore high amount ofsystems enzymes which only required heat the water step. to are are produced during to the germination a maximum of used 75 degrees and rely on high Grains to be for enzymatic purposes will water flow and minimal using a lot be germinated beforepressure, processing. Enzymeof active watermalt in the process. flour is produced from germinated Most wheat food related use hot of water barley, or rye. sites The functions these that comesare off the boiler through hoses, products generally similar. using Besides foamers, liquid chemicals and other alphaand beta-amylases, there sanitation agents. They between are other enzymes in can malt use flour, like pro200-1,000 litres of water While per hour to blast a teases and glucanases. some of these surface clean. They can also havesome the negahave positive effects on baking, do the tive side effect of sending dangerous bacteria opposite. Like the enzymes natively found airborne, through the plant in flour,spreading enzymes them from malt flour also have without killing them. This wet a considerable effect on the steam Falling system Number does not vapour process. (FN). If use the a enzymatic activity of flour is very low, malt flour up to 150 grams per 100 kg can be used order to decrease FN values Benefits of in dry steam cleaning to 250-300 seconds.capacity In the case FN below Efficient cleaning is of produced 300 the s, enzyme malt made flour may from steam active pressure on result the in sticky doughs even used at below surface to be cleaned andwhen the solvent power g per 100 at kg. of malt flour of 50 micro drops a The high activity temperature, with is mostly stated present. as the diastatic power (DP) minimal moisture and is continuous usually at around DP. This activity The steam400 system provides may besteam interpreted 80-120 constant quality as which can SKB/g. be adjusted by volume and dryness. Water flow and heating power can be controlled and adaptFungal amylase ed byUsually an electronic system. fungi control belonging to Aspergillus The amount of water saved depends upon genus are used to produce fungal amylase. the flow rate of the water system and the The species are well defined and do not pressure employed, it can save substances. up to 90 produce toxic orbut carcinogenic percent of water OspreyDeepcleans dry The fungi are used. grown in large fermentation steam will typically use between tankstechnology end left producing and excreting the 10-30 litres of water whereasfluid. a desired enzymes to per the hour, fermentation conventional system broth will use betweenthe 200The fermentation containing raw 1,000 litresis per hour. by centrifugation, filtraenzyme processed Conventional cleaning leaves the tion, precipitation, ultrafiltration andrisk theof like potentially residues contaminating to result dangerous in a purified concentrated enzyme food and raw materials. Then is you also haveto solution. This concentrate converted the issue ofby getting difficult spaces, such powder sprayinto drying, mixed with bulk as material tight crevices in storagethe areas or complex to standardise activity and free shaped equipment. flowing agents to provide ease of usage in Dry As steam sanitises surfaces, penetrating mills. a dry powder most enzymes are cracks, crevices and other hard-to-reach areas very stable and can be kept for years without where manual and traditional cleaning methods, a losing much activity. Figure 1 provides which mainly relyproduction on potentially hazardous scheme for the of enzymes from chemicals, fail to achieve the required standards. plants, animals and microbes.
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Figure 2: Hemicellulolytic enzymes Kopie

to any Amylase from fungal sources is mostly other cleanalpha-amylase. Most detrimental side activiing method. ties are prevented by selection of suitable No additional subspecies and processing conditions. At ingredients are required normal dosage, fungalin amylase does not the steam to improve cleaning interfere with FN values because it is not power, cleaning (approx. capacity 95 C) of stable as at efficient the temperature is the produced from the steam pressuremodistandard assay. There is another, made on test the to surface to be fied FN determine the cleaned effect of and fungal the solvent which power employs of micro drops a high amylase, lower at maximum temperature, with moisture temperatures at minimal around 82 C. present. However, The doswhere age of specific alphatasks or locaamylase tions demand preparations it, depends ingredients on can be added their activtoity. In improve order the to solidificaexpress tion the of specific activsubstances, for ity of alphainstance amylase,within the liquid unit fat for applithe cation devices test method which could developed congeal withby Sandstedt, out the use of Kneen and additional ingreBlish (1939), dients. the SKB, is used. Even though differDry steam ent suppliers machines use different for different test methods, applications the The results techare Osprey often nology converted has Deepclean into SKB. developed is For wheat available in a with of Falling range dry Numbers steam machines between 300 for different and 350 s, the applications, typical dosage based on many is adjusted as years experi500ofSKB per ence creating kg of steam flour bespoke (that is, This 1 solutions.

Hemicellulase
The term hemicellulase designates a family of enzymes. All the members shown in Figure 2 are able to break down the pentosans (therefore they are also called pentosanases), but their

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FEATURE impacts on dough and baking properties vary widely. It is assumed that pentosans form a network with gluten; the more pentosans are involved, the firmer the network. Because they bind large amounts of water (approximately 10 times their dry weight), they reduce the availability of water for the gluten reducing its expandability. Additionally, pentosans can be cross-linked to each other by the so-called oxidative gelation, increasing their ability to bind water further. That is a main reason why darker wheat flours and mixtures containing rye flour have a lower volume yield than white flours. The volume yield of all flours can be increased considerably by adding hemicellulases. Many of these enzymes are derived from Aspergillus strains selected for or specialising in the production of hemicellulases. Hemicellulases are often sold in compounds with amylase and other enzymes. The most common hemicellulase for baking applications is an endo-1, 4--xylanase. It is not possible to give a general dosage recommendation as there is no standard method of determining hemicellulase activity. The available methods are usually based on determining the release of reducing sugars, the reduction of viscosity or the breakdown of synthetic or coloured molecules and are very difficult to relate to each other. Moreover, even the use of a standard method for different hemicellulases does not necessarily permit conclusions in respect of baking properties, presumably because the points at which hemicellulases of different origin attack the pentosan molecules are too various.

Figure 3: Action of protease on gluten

Table 1: Enzymes suggested for bread and flour improvement (not exclusive) Enzyme Claimed Effect

Alpha-amylase, bacterial Alpha-amylase, cereal Alpha-amylase, fungal Alpha-amylase, maltogenic, intermediate heat stable Ascorbic acid oxidase Beta-amylase Branching enzyme (glucotransferase) Cellulase Furanosidase, arabinofuranosidase Ferulic and cumaric acid esterase Glucoamylase, (amyloglucosidase) Glutathion oxidase -glucanase Glucose oxidase, galactose oxidase, hexose oxidase Hemicellulase, xylanase, pentosanase Laccase, polyphenol oxidase Carboxyl esterase (lipase, phospholipase, galactolipase etc.) Lipoxygenase, lipoxidase Exo-Peptidase Peroxidase Protease, proteinase, endo-peptidase Pullulanase Sulfhydryl oxidase Sulfhydryl transferase Transglutaminase
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Oven-rise, anti-staling, liquefaction Oven-rise, anti-staling Energy supply for yeast, dough & bread structure Anti-staling Protein strengthening Energy supply for yeast, browning, taste Water binding Water binding Dough structure, water binding Dough structure, water binding Energy supply, colour, flavour Protein strengthening Structure, liquefaction Protein strengthening Dough structure, water binding, volume yield Dough strengthening Flavour, in-situ emulsification, dough stability and volume yield, dough brightening Dough structure, decolourization Colour, flavour Protein strengthening Protein relaxation, liquefaction Structure, water binding Protein strengthening Protein strengthening Protein cross-linking, gluten stabilization

Protease
Proteases (also known as proteinases or peptidases) split the protein strands of the gluten molecule (Figure 3) and thus lead first to a softening and then to a complete collapse of the structure. A purified single and very specific protease would only be able to break down a few of the peptide bonds, resulting in only limited softening. With short gluten structures a slight softening may well be desirable; in this case it has a similar significance to the use of L-cysteine. The proteolytic action is more time-dependant than the function of cysteine. As a result, it increases with the fermentation time of the dough. That is why there is a considerable demand for enzyme preparations that do not contain even traces of protease. The use of protease is less crucial with flours that are rich in gluten. It is even very common in the production of pan (toast) bread, where a soft dough that precisely fills the tin is required. Proteases are also very useful in the production of cracker,
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FEATURE such as the tyrosine groups in protein or the feruloyl residues in pentosans. The oxidative cross-linking of the pentosans is called oxidative gelation, a reaction resulting in increased dough firmness and dryer dough surfaces. The limiting factor in this process is the availability of oxygen because of other chemical and biochemical reaction consuming oxygen. Therefore, the conditions for oxidases are only good on the surface of the dough where plenty of oxygen is always available. This limitation can be solved by technical measures during dough preparation, for example overpressure or the supply of extra oxygen through the mixing tool.

Figure 4: Reaction of glucose oxidase and some probable effects on dough components

Carboxyl esterases
The term carboxyl esterase comprises all lipolytic enzymes, for example (triacyl) lipase, phospholipase and galactolipase. They all catalyse the hydrolysis of acyl residues (fatty acids) from lipids. Wheat contains about 2.5-3.3 percent lipids, a typical bread flour about 2.5-2.7 percent (Chung & Ohm, 2009), but only about 1 percent are free lipids that are easily accessible by lipolytic enzymes. A schematic representation of the lipids composition of wheat flour is given in Figure 5. Lipase converts non-polar lipids into the more polar structures diglycerides and monoglycerides, i.e. emulsifiers (Figure 6). Lipids of wheat flour are already polar to some extent, namely phospholipids and glycolipids are converted into more polar and hydrophilic lyso-forms by phospholipases and glycolipases. The in situ formation of mono- and diglycerides from wheat lipids results in dough strengthening and larger volume yield, but according to the authors findings doesnt have a significant effect on starch retrogradation and hence bread staling. This is in contrast to the effect of mono- and diglycerides which are added to a bread formula: due to interaction with starch they are able to reducing the staling rate. On the other hand, their effect on volume yield is very limited. Most probably, the action of enzymatically formed emulsifiers on volume yield is pronounced because they are already located at the right sites of the dough for improving the

Figure 5: Classification and distribution of the main lipids in wheat flour (averages; % d.s.; modif. from Pomeranz and Chung, 1978, using data from Chung and Ohm, 2009)

biscuit or wafer flours where elasticity of the gluten is not desirable.

Glyco oxidases
There are several oxidoreductases in nature that convert sugar molecules into the corresponding acids, or, as in the case of sorbitol oxidase, that convert a sugar alcohol into the corresponding sugar. The most common oxidase (from a commercial perspective) is glucose oxidase. Other examples are maltose or galactose oxidase. More generic terms used for all these enzymes are hexose oxidase or pyranose oxidase. The enzyme glucose oxidase (GOX) is usually derived from the mould Aspergillus, sometimes from Penicillium species. Honey

is also a rich source of GOX. The enzyme stems from the pharyngeal glands of the bees. However, its suitability is rather restricted by the taste of its carrier. One effect of GOX in the dough is to oxidize glucose to form gluconic acid with the aid of atmospheric oxygen, but the slight souring that occurs in the process is negligible; its other effect is to transform water into hydrogen peroxide (Figure 4). This oxidizing agent acts on the thiol groups of the gluten, either directly or via several pathways, inducing formation of disulphide bonds and thus tightening of the protein. Since hydrogen peroxide is a rather non-specific oxidizing agent, it may also react with other reducible substrates, for instance phenolic component

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FEATURE omitted; the dough pieces will keep the shape given by the cutting; shrinkage and bending in the oven as well as the formation of hairline cracks (checking) are avoided. With suitable amylases, expensive recipe components such as milk solids otherwise necessary for sufficient browning can be omitted. Furthermore, the whole process will be less dependent on flour quality.

Other flour applications

Enzymes have also been introduced into flour applications others than baking, for example noodles or steamed bread. For steamed bread the desired properties are similar to those in bread baking, but the technology is quite different and requires different types of enzyme compounds. In many types of steamed bread, specific lipases are very useful, providing stability, volume and a bright crumb colour. Except for certain types of Asian noodles, for example Japanese udon noodles, a firm bite and a high cooking tolerance are advantageous. For instant noodles, the requirements are quite different, because the reduction of oil uptake during frying and a fast water uptake upon cooking are of utmost importance. Furthermore, avoiding the cracking of dried noodles is a typical aim, although this problem is caused in many cases by inadequate drying conditions. Finally, the colour of the fresh or dry noodle is important. A bright colour without speckles is a desirable property of many but not all types of noodles. All of these challenges can be approached by enzymes, namely by carboxyl esterases and lipoxygenases.

Figure 6: Effect of carboxylesterases on wheat lipids

Figure 7: Cross linking of protein by transglutaminase

protein properties; but for anti-staling effects, not enough emulsifier is formed to interfere with starch retrogradation. Nevertheless, they have a distinct effect on the shelf life of bread because they create a better starting point at the beginning of the storage due to improved volume and crumb structure. With the staling rate unchanged, this results also in a better structure (crumb softness) at the end of the storage period. Interestingly, it is being disputed whether the doughs have to contain additional fat, and if so, what kind of fat, for the lipase to work satisfactorily. According to our findings, fat reduces the efficacy of lipase, probably by distracting the lipase from the right target, i.e. the flour lipids. Initially, there was also the problem of a possible impairment of taste due to the release of flavour-active fatty acids, particularly if butter is involved. The most recent carboxyl esterase are more specific in this concern and hence do not affect the flavour in most applications.

and glutamine groups to work. Although lysine is a limited amino acid in flour, the levels are enough for transglutaminase to work. The result is a strengthening effect on the dough, like ascorbic acid. Because it is rather expensive compared to ascorbic acid, its use is limited. A special usage area can be in very long or retarded fermentation processes where very low amounts of the enzyme will work sufficiently long.

References
Chung, OK, Ohm, JB, 2009. Wheat Lipids. In: Wheat - Chemistry & Technology, Khan, K, Shewry, PR (ed.), AACC Press, 363-399. Pomeranz, Y and Chung, OK, 1978. Interaction of lipids with proteins and carbohydrates in breadmaking. J. Am. Oil Chem. Soc., 285-289 . Sandstedt RM, Kneen E and Blish MJ, 1939. A standardized Wohlgemuth procedure for alpha-amylase activity. Cereal Chem. 16, 712-723.

Enzymes for biscuits, crackers and wafers

Whereas a high protein content and strong gluten are desired properties in many bread processes, flours with little and weak gluten are preferable for durable baked goods. The tendency of dough to spring back after rolling and the undesired formation of gluten lumps in wafer batter are the reasons for this requirement. Whether a flour with low and weak protein is available or not, the use of elasticity-reducing agents will have benefits in all stages of the process: The lamination will be more uniform; reduction of the thickness of the dough sheet can be performed faster and more reproducibly; relaxing periods for the dough sheet can be shortened or even

Read the second part of this article in the next issue of Grain and Feed Milling Technology. Lutz Popper will discuss additives other than flour and standardisation services.

Transglutaminase
This enzyme causes bond formation between protein folds or different protein strands (Figure 7). It needs lysine
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Additives for flour standardisation

Part I: Enzymes

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