Today we continue our discussion of enzymes, but this time we will explore some lesser-known enzymes, some of which have recently found applications in baking. You can refer to our previous blog post on enzymes in baking, where we covered general aspects and amylases, in the following link.
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Hemicellulases
Hemicellulases are now a common component in most bread improvers. It&#;s important to note that these enzymes can go by various names, such as pentosanases or xylanases. Hemicelluloses are non-digestible polysaccharides composed of more than one type of monomer. In the case of flours, these are usually xylose, arabinose, and galactose. These monomers are linked by β-1,4 bonds, which are challenging to hydrolyse in the intestinal tract. Nutritionally, they are considered dietary fibres. Since the monomers forming hemicelluloses in wheat flour are pentoses, they are sometimes referred to as pentosans, which is why the enzymes capable of hydrolysing them are called pentosanases. Additionally, the most common pentosans in bread dough are arabinoxylans, which consist of a chain of xyloses with added arabinose branches. Enzymes that can break the xylose chain are known as xylanases, making them a specific type of hemicellulase or pentosanase.
Wheat flour contains a small percentage of hemicelluloses, around 4%. These compounds can be either soluble or insoluble and have a high water-absorbing capacity. In fact, they can absorb nearly a quarter of the water in the dough. Enzymes that hydrolyse these compounds reduce their water-absorbing capacity, releasing water that becomes available to hydrate other components of the dough, such as starch and proteins. When these enzymes are added, the dough becomes less consistent and tenacious, and more extensible, improving its workability, or its ability to be rolled out and shaped. The enhanced extensibility leads to better dough expansion during fermentation and in the early stages of baking. Moreover, these enzymes do not appear to have negative effects on the dough.
In dough made with high percentages of rye flour, the use of these enzymes is particularly important because rye flour contains more arabinoxylans than wheat flour. In such cases, these enzymes are essential for a proper baking process.
Lipases
Although the lipid content in dough is low, lipases, enzymes capable of hydrolysing lipids, can be beneficial in the baking process. Lipids are composed of triglycerides, which, in turn, consist of glycerol and three fatty acids. Lipases work by cleaving these fatty acids. Cutting one of them generates a diglyceride, and cutting two produces a monoglyceride. Most lipases cut the two outermost fatty acids, leaving the middle one intact, thereby producing monoglycerides and free fatty acids. As discussed in the section on emulsifiers, monoglycerides are substances that effectively reduce starch retrogradation and slow down bread staling. Thus, the use of lipases can extend the shelf life of bread without the need for additives, reducing the presence of additives both in the labelling and in the final product.
While early lipases function as described, a new generation of lipases naturally produces products with a similar function to DATEM, an emulsifier that interacts with gluten to enhance dough. These lipases belong to the group of phospholipases, which hydrolyse phospholipids. Like their predecessors, these newer lipases reduce the need for emulsifiers and, consequently, additives. However, while the first group has an anti-hardening effect, the latter enhances dough strength.
The fatty acids generated by lipases can also play a role in the aroma and flavour of the products. In fact, this flavour effect is the basis of their use in cheese production.
Proteases
Proteases are enzymes that hydrolyse proteins. In the context of bread dough, they act by breaking down gluten, reducing its strength. While proteases are not commonly used in bread baking in Spain, they find applications in other countries where stronger flours are used. Proteases can help reduce dough mixing times. They also make the dough more flowable for expansion within a mold and can be useful in other preparations. Overall, the use of proteases has a similar effect to that of reducing agents, which relax the dough by breaking disulfide bonds. However, there are two significant differences. First, the action of reducing agents is reversible, whereas protease-induced protein breakdown is irreversible. Second, while reducing agents have a quick effect at the beginning of mixing, proteases work more slowly, depending on the time and temperature of the dough.
To shorten mixing times effectively, proteases should be added during sponge fermentation, which is then mixed with the rest of the flour. This allows the proteases sufficient time to act, but they do not affect the entire dough. Proteases are also being explored for use in products that need to expand within molds, such as hamburger or hot dog buns, as they make the gluten network less elastic, facilitating lateral expansion. However, the action of proteases on gluten during fermentation can impair its gas-retention capacity, resulting in dough collapse during fermentation or at the beginning of baking.
Proteases can also be useful in making saltine crackers or pizza dough, as they reduce dough elasticity and increase its extensibility. However, excessive proteolysis can be harmful.
A new application of proteases involves improving crusts. Hydrolysing proteins in the crust results in more free amino acids and enhances Maillard reactions, producing darker crusts. What&#;s less known is that the hydrolysis of proteins in the crust also helps create crisper crusts. If this effect is desired, the protease should be applied to the dough&#;s surface, not incorporated into the formulation, to avoid degrading the gluten network responsible for gas retention. This application can be valuable in crusty breads production but not in sandwich bread, where the crust should not be crisp.
Among proteases, those acting inside proteins, breaking bonds between amino acids randomly (endo-), are preferred over those working at the ends, cleaving bonds one by one (exo-). The latter have a much smaller effect on dough strength, but they can be used in biscuit production. Additionally, there are different proteases based on their optimal pH and specificity in breaking specific amino acid bonds. Therefore, it&#;s crucial to choose the right type of protease as not all of them work the same.
Glucose Oxidase
Glucose oxidase is classified as a dough strengthener enzyme. Its function involves converting glucose into gluconic acid and hydrogen peroxide. This enzyme has been used in the production of powdered eggs to reduce glucose content, preventing it from darkening products through Maillard reactions with amino acids from egg proteins. In bread dough, what&#;s primarily interesting is the generated hydrogen peroxide, a powerful oxidizer. This action strengthens the dough by promoting the formation of disulfide bonds in the gluten network. Therefore, the use of glucose oxidase can partially or entirely replace additives like ascorbic acid. Initially, it was thought that the low glucose levels in dough could limit the effect, and that it might be necessary to increase glucose levels using glucoamylases. However, it was found that the amount of glucose produced by the amylases present in the flour is sufficiently significant.
Transglutaminase
Transglutaminase is an enzyme capable of binding lysine and glutamine, both amino acids found in proteins. This enzyme has been widely used in meat products and fish derivatives to bind these proteins and create meat-like products (restructured meats) from leftover meat scraps adhering to bones. Transglutaminase has also been used as a coadjutant in the production of surimi (seafood analogues made from more economical raw materials). In the case of bread, the low lysine content and the initial high cost of the enzyme delayed its inclusion in dough formulations. Nevertheless, early research showed that the lysine present in wheat proteins is sufficient for transglutaminase to have a pronounced effect.
By binding proteins, transglutaminase has a clear strengthening effect on dough. However, this effect results in dough that is very tenacious, less extensible, and quite dry and challenging to work with. For this reason, transglutaminase has not been widely incorporated into bread improvers. It is possible that, with proper dosing or when combined with other additives or enzymes, it might have a positive impact, but more convenient alternatives exist.
Transglutaminase has also been tested for binding proteins in the production of gluten-free products, but it is incapable of forming a protein network similar to gluten, so it usually is not used in these types of preparations.
Lipoxygenase
Traditionally, soybean flour, more common in North America, or broad bean flour, more prevalent in Europe, has been used as an ingredient in baking. This practice is due to the presence of lipoxygenase in these flours. Lipoxygenase is an enzyme capable of oxidizing specific fatty acids, such as linoleic or linolenic acid, as well as carotenoids and chlorophylls, which are colouring substances. Some lipoxygenases have a more pronounced bleaching effect as they oxidize colouring substances, while others have a more significant dough-strengthening effect by oxidizing fatty acids.
The bleaching effect of lipoxygenases can be a drawback when aiming for a more yellowish colour, such as in pasta production. In fact, for pasta, it is preferable to use durum wheat varieties with low lipoxygenase content and avoid air incorporation during mixing to reduce the effect of lipoxygenases. However, this effect is beneficial when desiring whiter colours. In English-speaking countries, a whitish colour is an important quality factor for bread, making this enzyme highly useful. In Spain, this parameter is less important, and we are accustomed to slightly creamy bread crumb colour. It is more valued in sandwich bread, where this ingredient has traditionally been used.
The oxidation of fatty acids produces substances that enhance tolerance to excessive mixing and strengthen dough by improving the retention of gases generated during fermentation. This effect is related to the enhancement of disulfide bonds in the gluten network but is still not fully explained. For this effect to occur, the presence of oxygen and fatty acids is necessary. Therefore, it happens during mixing, where air is incorporated into the dough, and in the early stages of fermentation. The action of these enzymes is more potent in bread formulations containing a small proportion of oil. For this reason, its use is more frequent in sandwich bread and similar products.
Commercial lipoxygenases are now available, eliminating the need for soy or broad bean flour. It appears that the effect of these enzymes is more dough-strengthening than bleaching. It is also important to note that the oxidation of fatty acids can generate rancid flavours. This effect is more significant in bread with lean formulations but is less noticeable in products containing flavour-rich ingredients like milk, sugar, seeds, or whole-grain flours.
Asparaginase
Asparaginase is a recently introduced enzyme in baking. This enzyme is capable of hydrolysing asparagine, an amino acid present in proteins, into aspartic acid and ammonia. The interest in using asparaginase in baking lies in the fact that this reaction can significantly reduce acrylamide levels. Acrylamide is a compound produced in the Maillard reaction and has been shown to be carcinogenic. Concerns about acrylamide levels in food arose in after a study by Swedish researchers. Acrylamide is primarily produced through the reaction between asparagine and reducing sugars under conditions of heat and low humidity, such as those found in bread crust during baking. Therefore, the hydrolysis of asparagine by asparaginase can reduce acrylamide levels in the final product.
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This study was carried out to evaluate the effects of amyloglucosidase, glucose oxidase, hemicellulase (mainly consist of endo-1,4-β-xylanase), cellulase, lipase, and the combination of phospholipase and hemicellulase (phospholipase + hemicellulase) on the extensographic properties of dough and the quality characteristics of bread prepared from wheat meal. The enzymes were added separately in two different amounts. The addition of glucose oxidase (at 0.&#;0.001%) caused a significant decrease in the resistance to extension, ratio of resistance to extensibility and energy values of the wheat meal dough compared with the control dough. The addition of hemicellulase (at 0.001&#;0.005%) and phospholipase + hemicellulase (at 0.&#;0.%) also improved the wheat meal dough rheology by reducing the resistance to extension and the ratio of resistance to extensibility. Glucose oxidase (at 0.&#;0.001%), hemicellulase (at 0.001&#;0.005%) and phospholipase + hemicellulase (at 0.&#;0.%) addition improved the specific volume of wheat meal bread compared with the control bread. Increasing the dosage of glucose oxidase from 0. to 0.001% caused a further increase in the specific volume of wheat meal bread. The addition of hemicellulase (at 0.001&#;0.005%) caused a significant decrease in the baking loss and an increase in the moisture content of wheat meal bread compared with the control bread. The addition of amyloglucosidase (at 0.&#;0.001%), lipase (at 0.&#;0.001%) and cellulase (at 0.&#;0.%) did not considerably affected the dough rheological and the quality characteristics of wheat meal bread.
The main objective of our study was to evaluate the effects of the addition of commercial amyloglucosidase, glucose oxidase, hemicellulase (mainly consist of endo-1,4-β-xylanase), cellulase, lipase and the combination of phospholipase and hemicellulase (phospholipase + hemicellulase) on the extensographic properties of dough and the quality characteristics of bread prepared from WM.
Starch and proteins are the major constituents of wheat flour and they affect the rheological properties of dough and consequently the characteristics of the final baked product (Kaur et al. ). Siddhartha Kumar et al. ( ) defined that glucoamylase (amyloglucosidase) hydrolyzes both 1,4-α- and 1,6-α-glucosidic linkages and releases β-d-glucose from the non-reducing ends of starch and related oligo- and poly-saccharides. Glucose oxidase is an oxidative enzyme that catalyzes the oxidation of β-d-glucose to gluconic acid and hydrogen peroxide. In breadmaking, glucose oxidase induces the formation of disulfide bonds in gluten proteins. The use of the proper dosage of glucose oxidase improves dough properties and bread quality (Bonet et al. ). The minor wheat flour constituents, e.g. non-starch polysaccharides, such as arabinoxylans (AX) and β-glucans, also play a significant role in the breadmaking process, affecting the properties of the dough and the final baked product. AX are classified as water extractable AX (WE-AX) and water unextractable AX (WU-AX) (Courtin and Delcour ). Courtin et al. ( ) indicated that WU-AX are detrimental to breadmaking while WE-AX with medium to high molecular weight have a positive impact on bread volume (Shah et al. ). Hemicellulases, which hydrolyze pentosans, and cellulases, which hydrolyze complex cell wall carbohydrates, improve the handling properties of dough and bread quality (Harada et al. ). Among hemicellulase enzymes endo-xylanases and β-xylosidases are the two key enzymes responsible for the hydrolysis of xylan, the major component of hemicellulose (Juturu and Wu ). Xylanases are able to hydrolyze the xylan backbones of WU-AX releasing WE&#;AX, which have positive effects on dough and bread characteristics (Courtin and Delcour ). Lipids and phospholipids are also minor components of wheat kernel. Lipases hydrolyze triglycerides into monoglycerides, diglycerides, fatty acids and glycerol. Lipases improve volume, texture and shelf-life of bread (Hasan et al. ). Phospholipases convert phospholipids into fatty acids and other lipophilic substances (Salehifar et al. ). Phospholipases provide the dough with a suitable degree of elasticity and extensibility, and also improve bread volume (Néron et al. ).
Various studies have been conducted to modify dough rheology and bread quality. Grosch and Wieser ( ) indicated that ascorbic acid is used as a bread improver to improve bread volume and crumb structure. Miyazaki et al. ( ) have examined the effects of dextrins on bread quality and dough properties. Gómez et al. ( ) reported that some emulsifiers, like sodium stearoyl lactylate, diacetyl tartaric acid esters of monoglycerides and polysorbate exhibit their positive effects during the mechanical handling and proofing of dough. Emulsifiers also improve bread volume and crumb structure. However, today, one of the most common alternatives in breadmaking to improve dough properties and bread quality, is the use of enzymes.
Bread is one of the oldest forms of prepared foods and it has been a staple source of nutrients for humans for several thousands years. As bread is one of the major constituents of the human diet, wheat is by far the most important cereal in breadmaking (Geng et al. ). The most of cereal products are traditionally prepared from wheat flour (WF). However, the endosperm, germ and bran parts of the wheat kernel are separated during the milling processes which causes the loss of many health beneficial micronutrients, minerals, and fibre. Whole wheat flour is one of the most common and important whole grain products. It is a rich source of dietary fibre, vitamins, minerals, and antioxidants (Hirawan et al. ). Epidemiological studies have reported that the long-term intake of whole grain products can reduce the risk of several chronic diseases, such as diabetes, cardiovascular diseases and cancer (Vitaglione et al. ). Therefore, as public awareness of eating healthy foods grows, whole grains and whole grain products, such as wheat meal (WM), are increasingly used in the production of many kinds of cereal based foods. Despite the fact that foods containing whole grain products have health beneficial effects, the production of these foods in good quality is very challenging from the standpoint of consumers&#; acceptance. Gan et al. ( ) reported that the public&#;s acceptance of whole wheat food products is limited due to their poor taste and texture. As consumers&#; preferences are shifting towards the consumption of healthier foods, the bakery industry is focused on the production of food products containing whole grain products in good quality.
Breadmaking trials were performed in duplicate. Four bread samples were obtained at each trial and the best two of four bread samples in terms of shape uniformity from each trial were chosen for bread quality assessment. Chosen breads were cooled for 2 h at 25 ± 2 °C and then weighed before quality analyses. Quality characteristics of breads were assessed in terms of specific volume (SV, cm 3 /g), baking loss percentage (BL, %) and moisture content (MC, %). Loaf volume was measured according to the rapeseed displacement method using a mechanical volumeter. SV was calculated as the ratio between the volume and the mass of the bread. BL was calculated as a percentage ratio of the mass of water removed from the dough during baking and the mass of dough before baking. MC was determined according to AACC International Method 44-15A ( ). Bread quality analyses&#;comprised of SV, BL and MC analyses&#;were conducted in four replicates for each chosen bread sample. The mean values of the results of the analyses were used in statistical analyses.
The breadmaking procedure was carried out according to the procedure described in the literature (Standard-Methoden für Getreide Mehl und Brot. 6, , Verlag Moritz Schafer, Detmold, Germany) with slight modifications. In breadmaking trials, 100% flour (WM and WF), enzyme (when added), 1.2% salt, 3% compressed yeast (based on flour weight at 14% moisture), water at 30 ± 1 °C (according to the WA determined by the farinograph) were kneaded using a laboratory scale L-shaped mixer (Diosna Dierks & Söhne GmbH, Germany) at 100 rpm for 7 min (we determined the optimal kneading time by pre-studies). At the end of kneading, the leavening step was performed twice using a proofing cabinet (at 32 ± 2 °C and 75&#;80% relative humidity) each for 30 min. The dough was aerated in the mixer for 1 min at the end of each leavening step. After the second (last) aeration step, the dough was divided into 500 g pieces, hand rounded, molded and proofed for 45 min at 32 ± 2 °C and 75&#;80% relative humidity. Baking was carried out for 35 min at 220 °C in a ventilated oven (Özköseoğlu Rotatherm Oven, Turkey) following a steam injection at the beginning.
Extensograph tests were carried out according to ICC Standard Method Nr. 114/1 ( ) using the extensograph (Mod. FXEK/6 Nr. 466, Brabender, Duisburg, Germany). The parameters obtained from the extensograph curves were resistance to extension up to 50 mm (R 50 , BU), extensibility (E x , mm), ratio of resistance to extensibility (R 50 /E x ) and energy (A e , cm 2 ). The farinograph and extensograph tests were performed in duplicate.
Moisture content was determined according to ICC Standard Method Nr. 110/1 ( ) using a laboratory scale oven (Köttermann Typ , Germany). Ash content was determined according to ICC Standard Method Nr. 104/1 ( ) using an ash oven (Carbolite, Type: ELF 11/68, England). Protein content was determined according to AACC International Method 46-12 ( ) using an automatic distillation system (Gerhardt Vapodest 20, Type VAP 20, Germany). The total and soluble pentosan contents were determined according to the method stated in Hashimoto et al. ( ) using an UV&#;visible spectrophotometer at 670 nm (Varian Cary 50 Bio UV&#;visible Spectrophotometer, USA). Damaged starch content was determined according to McDermott ( ) using the UV&#;visible spectrophotometer at 600 nm. Wet gluten quantity was determined according to ICC Standard Method Nr. 155 ( ) using a gluten washer apparatus (Yücebaş Makine, Type 111, Turkey). α-amylase activity was determined according to ICC Standard Method Nr. 107/1 ( ) using a Falling Number apparatus (Falling Number AB S-, Type , Sweden). Chemical composition analyses of WM and WF were performed in triplicate. All chemicals and laboratory reagents used in this study were analytical grade.
Commercial type WM and WF were supplied from a commercial miller in Turkey. The following enzymes used in this study were provided from an enzyme supplier in Turkey, which imported the enzymes from an enzyme manufacturer in Europe: amyloglucosidase (AGL; standardized activity: 65,000 AGI/g), glucose oxidase (GOX; standardized activity: SRU/g), hemicellulase (HML; mainly consist of endo-1,4-β-xylanase; standardized activity: EDX/g), cellulase (CLL; standardized activity: 25,000 CXU/g), lipase (LPS; standardized activity: 80,000 PLI/g), and the combination of phospholipase and hemicellulase (PHM; standardized activity: DLU/g). AGL, GOX, HML, CLL, LPS and PHM were added to WM separately (not in combinations) at two different dosages, according to the enzyme supplier&#;s recommendations. The enzyme dosages added to WM were: AGL = 0. and 0.001%; GOX = 0. and 0.001%; HML = 0.001 and 0.005%; CLL = 0. and 0.%; LPS = 0. and 0.001%; PHM = 0. and 0.% (w/w, flour weight basis). WF, WM and enzyme added WM were mixed for 1 h using a mixing machine (Type LDK M73, Apparatebau JEL J. Engelsmann AG, Ludwigshafen/Rhein, Germany) before analyses.
Chemical composition data of WM and WF are presented in Table .
The ash content of WM was found to be higher than WF because of the higher bran content of WM. This was in agreement with the findings of Bruckner et al. () who found that whole meal has higher ash content compared with WF. In the present study, the ash content of WF was found to be 0.62%, which is in agreement with the findings of Sakhare et al. () who found 0.54&#;0.95% ash contents for straight run flours and bran duster flours. McCleary () reported that whole meal contains 5% and WF 2.5&#;3% of hemicellulose. In our study, total pentosan contents of WM and WF were found to be 6.37 and 1.35%, respectively. The total, soluble and insoluble pentosan contents of WM were found to be higher than WF, which was related with the high amount of bran content of WM. Damaged starch contents of WM and WF were found to be between 5 and 6% with no remarkable differences (P < 0.05). The α-amylase activity was inversely correlated to the falling number thus a low value of this parameter corresponded to a high α-amylase activity. In the present study, the falling number of WM (211.3 s) was found to be lower than WF (343.7 s). The falling number of WF was in agreement with the findings of Sakhare et al. () who found 301.3&#;396.3 s falling numbers for straight run flours and bran duster flours.
The farinographic properties of non-enzyme added WM and WF are presented in Table . The addition of AGL, GOX, HML, CLL, LPS and PHM to the WM presented slight and negligible changes in the farinographic properties. Therefore, the farinographic properties of AGL, GOX, HML, CLL, LPS and PHM added WM are not presented.
As seen in Table , WA of WM was found to be higher than WF as expected. This was in agreement with the findings of Bruckner et al. () who found that whole meal has higher WA compared with WF. Finney et al. () reported that WA of flour was affected by the protein content, damaged starch content and the non-starch carbohydrates content of flour. In the present study, the increase in WA of WM compared with WF was most probably related with the higher bran content consequently the higher total pentosan content of WM. Schmiele et al. () pointed out that the addition of whole wheat flour or wheat bran to WF causes an increase in the bran content of flour blend leading to an increase in WA (Bucsella et al. ).
DT and ST of WM dough (WMD) were found to be higher than WF dough (WFD). This was in agreement with the findings of Sakhare et al. () who found that DT and ST of bran duster flours (high extraction rate flours) were higher than straight run flours. Kaur et al. () reported that dough strength (in particular DT and ST) is not positively related to the protein content of flours, from which it can be inferred that flours with higher protein content may not necessarily have higher dough strength. In a study conducted by Chaudhary et al. (), it was noticed that the DT and ST increased in wheat varieties as per the quantity of glutenin in their composition. This was attributed to the intermolecular disulfide bonding between the glutenin polypeptides and formation of long chain polymers exhibiting a stronger network in dough. In the present study, the increase in DT of WMD compared with WFD may be attributed to the effect of the interaction between fibre and gluten (Rosell et al. ). Since bran particles were able to absorb high amount of water during mixing, protein hydration was prevented; consequently, the DT of dough increased. Mudgil et al. () found that DT of dough increased significantly with the addition of partially hydrolyzed guar gum to wheat flour. The increase in levels of partially hydrolyzed guar gum fortification caused an increase in DT of dough may be due to higher WA of partially hydrolyzed guar gum. They also found that a remarkable increase in DS of dough with the addition of partially hydrolyzed guar gum at 4 and 5% levels. In our study, DS of WMD was found to be lower than WFD which was similar to the findings of Rosell et al. () found that ST showed negative correlation with DS for fibre enriched flour.
The extensographic properties of enzyme added WMD, non-enzyme added WMD (WMDC) and WFD at 45, 90 and 135 min resting time periods are presented in Table .
R50 and R50/Ex of WMDC were found to be higher than WFD, and Ex and Ae of WMDC were found to be lower than WFD, throughout 135 min resting time period (Table ). The high amount of bran present in WM, in particular the high amount of non-starch polysaccharides content in WM could most probably cause an increase in R50 and R50/Ex, and a decrease in Ex and Ae of dough. This was similar to the findings of Miś et al. () who reported that the interactions between gluten matrix of the dough and carob fibre were strong enough to counteract the weakening effect of hydration on dough structure which resulted in an increase in resistance to extension of dough containing carob fibre. Courtin et al. () pointed out that resistance to extension of dough increased and extensibility of dough decreased with increase in WU-AX content of flour (Courtin and Delcour ). Water unextracted solids, mainly containing WU-AX negatively affect the dough properties because they have a higher water binding capacity, which was reflected in a higher maximum resistance to extension and a smaller extensibility at maximum resistance to extension of dough (Wang et al. ).
R50 of WMDC, WFD and enzyme added WMD increased with the increase in resting time from 45 to 135 min (regardless 90 min). At 135 min resting time, the highest R50 was found in WMD + AGLH (691 BU). Diler et al. () indicated that amyloglucosidase uses a part of the water during mixing, slowing down dough hydration. At 135 min resting time 0. and 0.001% GOX added WMD had the lowest R50 among all WMD samples tested in our study. Also, no significant difference was observed between R50 of GOX added WMD and WFD (P < 0.05). In Table , it is apparent that R50 of 0. and 0.001% GOX added WMD (447 and 471 BU, respectively) is very close to R50 of WFD (458 BU). These findings led to the conclusion that the addition of GOX causes a considerable decrease in R50 of WMD. This was most probably be related with the hydrogen peroxide produced during glucose oxidase reaction. The hydrogen peroxide promotes a weakening effect on the glutenin network structures, and in consequence the modification of dough viscoelastic properties (Bonet et al. ). However, increasing dosage of GOX from 0. to 0.001% did not cause a further decrease in R50 of WMD. The addition of AGL at 0.%, HML at 0.001&#;0.005%, CLL at 0.%, LPS at 0.001% and PHM at 0.&#;0.% also caused a significant decrease in R50 of WMD compared with WMDC at 135 min resting time (P < 0.05). Primo-Martín et al. () indicated that xylanases soften the dough structure, which is similar to our findings. The addition of CLL at 0.% and LPS at 0.% did not show any significant effects on R50 of WMD compared with WMDC at 135 min resting time (P < 0.05).
As expected, WFD had the highest Ex values throughout 135 min resting time among all the dough samples tested. Ex of WMDC and enzyme added WMD samples decreased with the increasing resting time from 45 to 135 min (regardless 90 min). However, no significant difference (P < 0.05) was observed between Ex of WMDC and enzyme added WMD samples at 135 min resting time, respectively (Table ). At 135 min resting time, although not statistically significant (P < 0.05), Ex of 0.005% HML and 0.% PHM added WMD was found to be higher than all WMD samples tested in the study. Rouau et al. () reported that xylanases led to moderate release of water initially absorbed by WU-AX, which caused a redistribution of water that became available for gluten leading to optimal development. Solubilisation of AX plays a positive role in dough rheology, improving the extensibility and gas retention of dough (Jiménez and Martínez-Anaya ). Phospholipases also provide the dough with a suitable degree of elasticity and extensibility (Néron et al. ). However, although not statistically significant (P < 0.05), Ex of 0.% PHM added WMD was found to be lower than Ex of 0.% PHM added WMD in the present study.
The highest R50/Ex was found in WMDC and 0.001% AGL added WMD, and the lowest R50/Ex was found in WFD at 135 min resting time among all the doughs tested in the study (Table ). The addition of GOX (at 0. and 0.001%), HML (at 0.005%) and PHM (at 0. and 0.%) caused a significant decrease in R50/Ex of WMD compared with WMDC at 135 min resting time (P < 0.05). The highest Ae was found in WFD throughout 135 min resting time period. At 135 min resting time, Ae of 0. and 0.001% GOX added WMD was found to be lowest among all WMD doughs tested in our study. This finding led to the conclusion that the energy required for handling of WMD reduces with the use of GOX.
Lipases are used in breadmaking to improve dough and bread characteristics (Moayedallaie et al. ). However, the addition of LPS at 0. and 0.001% did not considerably affect the extensographic properties of WMD, in our study. Underkofler () pointed out that lipase activity in flour for baking could be undesirable because free fatty acids have a detrimental effect on dough (Lin ). In the present study, the addition of CLL at 0. and 0.% did not considerably affect the extensographic properties of WMD.
SV values of breads are presented in Fig. , BL and MC values of breads are presented in Table . WMB represents WM bread, WMBC represents non-enzyme added WM bread, WFB represents WF bread.
Open in a separate windowLoaf volume is one of the most important measures of bread quality (Bruckner et al. ). It is well known that the volume of bread produced from WM is lower than the volume of bread produced from WF. In the present study, volume values of all WM breads were found to be significantly lower than WF bread (P<0.05). This was in agreement with the findings of Bruckner et al. () who found that whole meal bread has lower loaf volume compared with the volume of bread produced from WF. Noort et al. () pointed out that the bread produced from whole grain flour showed reduced loaf volume (Bucsella et al. ). This was explained by the mechanical disruption of the gluten network by hard bran particles in dough containing high amount of bran (Gan et al. ). Lai et al. () reported that bran added white flour binds large volume of water and so gluten is not properly hydrated. Poorly hydrated gluten causes lower loaf volume (Bruckner et al. ). Mudgil et al. () found that the loaf volume decreased with the addition of increasing levels of partially hydrolyzed guar gum. This was attributed to the diluting effect of gluten caused by partially hydrolyzed guar gum addition. Ognean et al. () indicated that the WU-AX present in bran reduce the gas retention capacity of dough and volume of bread. Protein content and amylase activity of flour also played an important role on the loaf volume. Chaudhary et al. () found that loaf volume was positively influenced by protein content. Cauvain and Young () pointed out that flour with low falling number produced weaker dough and lower loaf volume (Chaudhary et al. ).
Specific volume which comprises loaf volume and loaf weight is an important parameter to analyze the quality of bread (Mudgil et al. ). Since a remarkable decrease or increase in specific volume is due to the strength of the gluten network, specific volume can be elucidated as the potential of gluten network to entrap CO2 produced during proofing in breadmaking (Chaudhary et al. ).
As seen in Fig. , SV of WFB (2.51 cm3/g) was found to be significantly higher than WMBC (1.66 cm3/g) and enzyme added WMB samples (varied between 1.57 and 1.83 cm3/g), in the present study (P < 0.05). This result was an expected result because the total pentosan content of WM was found to be higher than WF, and the protein content and falling number values of WM were found to be lower than WF. This was also in agreement with the findings of Miś et al. () who reported that fibre-rich breads were characterized by reduced volume.
In the present study, the addition of GOX at 0.001% significantly improved SV of WMB compared with WMBC (P < 0.05). Although not statistically significant, SV of 0.% GOX added WMB was found to be higher than SV of WMBC (P < 0.05). Additionally, the increasing dosage of GOX from 0. to 0.001% caused a further increase in SV of WMB. These findings indicate that the use of proper dosage of GOX improves the volume of WMB. This effect is most probably related with the positive effects of GOX on WMD rheology (Table ).
Courtin and Delcour (), Martínez-Anaya and Jiménez (, ) and Rouau et al. () indicated that the utilization of xylanases exhibits positive effects in baking. Shah et al. () reported that the addition of xylanases increased SV of WMB, significantly. In our study, the addition of HML at 0.001 and 0.005% significantly improved SV of WMB compared with WMBC (P < 0.05). Ognean et al. () indicated that the conversion of WU-AX in WE-AX by xylanases has positive effects on bread characteristics. Gan et al. () reported that WE-AX form highly viscous solutions in dough aqueous phase which they increase the stability of the liquid films surrounding gas cells. This phenomenon prevents the gas diffusion from dough and increases the gas retention capacity of dough (Courtin and Delcour ). Therefore, volume of bread increases. Although not statistically significant (P < 0.05), SV of 0.005% HML added WMB was found to be lower than SV of 0.001% HML added WMB, in the present study.
Néron et al. () reported that phospholipases provide the dough with a suitable degree of elasticity and extensibility resulting in an increase in bread volume. In our study, the addition of PHM (at 0. and 0.%) significantly improved SV of WMB compared with WMBC (P < 0.05). Although not statistically significant (P < 0.05), SV of 0.% PHM added WMB was found to be lower than SV of 0.% PHM added WMB.
In the present study, WMB samples containing 0.% AGL and 0.% LPS exhibited lower SV values than WMBC, significantly (P < 0.005). Free fatty acids and other lipid degradation products produced during lipase reaction in dough can act as foam destabiliser, which causes reduced bread volume (Gan et al. ). The addition of 0.001% AGL and 0.001% LPS did not cause any significant differences in SV of WMB samples compared with WMBC. Although not statistically significant (P < 0.05), SV of 0.001% AGL added WMB was found to be higher than SV of 0.% AGL added WMB. Similarly, SV of 0.001% LPS added WMB was found to be higher than SV of 0.% LPS added WMB. The addition of 0. and 0.% of CLL did not significantly improve SV of WMB compared with WMBC (P < 0.05). However, although not statistically significant (P < 0.05), SV of 0.% CLL added WMB was found to be higher than SV of WMBC.
During baking, when dough is transformed into bread, one of the most significant phenomena is moisture loss. If too much moisture is lost during the baking, the product become underweight. In addition, water loss during the baking process has disadvantageous effects on the freshness of baked get staled (Kotoki and Deka ). In our study, all WMB samples exhibited higher BL than WFB (Table ). This was most probably related to the higher WA of WM than WF (Table ). The BL of WFB and WMBC was found to be 8.42 and 13.48%, respectively. The BL of enzyme added WMB samples varied between 10.91 and 14.51%. The BL of 0.001 and 0.005% HML, and BL of 0. and 0.001% AGL added WMB samples were found to be significantly lower than WMBC (P < 0.05). Among all WMB samples, the lower BL values were observed in WMB samples containing 0.001 and 0.005% HML. Our findings on BL of HML added WMB samples are most probably related to highly viscous solutions in the dough aqueous phase formed by WE-AX with the addition of HML. We assumed that highly viscous solutions in the dough aqueous phase prevented the release of water during baking. The BL of WMB samples containing 0.% CLL and 0.% PHM was found to be significantly higher than WMBC (P < 0.05). Among all WMB samples, the highest BL values were observed in WMB samples containing 0.% CLL and 0.% PHM. It should be noticed that the second highest BL was observed in WMB containing 0.% CLL among all WMB samples. We assumed that the water absorbed by bran particles in WMD could be released by the hydrolyzation of complex cell wall carbohydrates with the addition of CLL, from which more water was removed from dough during baking. Therefore, the BL values of CLL added WMB samples were found to be higher than WMBC, in our study.
MC of WFB and WMBC was found to be 37.08 and 38.85%, respectively. MC of enzyme added WMB samples varied between 38.06 and 40.53%. The peak values of MC were observed in WMB samples containing 0.001 and 0.005% HML. This finding is most probably related with the formation of highly viscous solutions in the dough aqueous phase with the addition HML. The formation of highly viscous solutions in the dough aqueous phase prevents the release of water during baking.
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