FUNGAL PHYTASE AS A POTENTIAL BREADMAKING ADDITIVE
Monica Haros*, Cristina M Rosell, Carmen Benedito
Laboratorio de Cereales, Instituto de Agroquímica y Tecnología de Alimentos (CSIC), P.O. Box 73, 46100 Burjassot, Valencia, Spain *
Departamento de Industrias y Química Orgánica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria, 1428 Capital Federal, Argentine
Correspondence should be addressed to: Cristina M Rosell Laboratorio de Cereales Instituto de Agroquímica y Tecnología de Alimentos (CSIC) P.O. Box 73, 46100 Burjassot, Valencia, Spain Tel: 34 96 390 00 22 Fax: 34 96 363 63 01 e-mail: [email protected]
ABSTRACT Phytase is a natural occurring enzyme in cereal flours and derived products, that catalyzes the hydrolysis of phytates resulting in a decrease of the phytate content (considered as antinutritional compound). However a high content of phytate is still present in the cereal derived products. The effect of the addition of exogenous phytase in four different bread formulations containing fiber on the breadmaking process will be analyzed. In all the formulations tested, the supplementation of phytase promoted an acceleration of the fermentation process; and at the fresh bread level, an improvement in the shape, a light increase of the specific volume, and also better crumb (softness effect) were obtained. Additionally, it is important to remark that the phytate content in doughs and fresh breads was reduced by the addition of phytase, with the subsequent nutritional benefits that this implies as consequence of reducing the anti-nutrient content of the breads containing fiber.
Keywords: phytase, phytate, breadmaking, bread quality
INTRODUCTION Currently, the health authorities world-wide recommends an increase in the consumption of whole grain cereal products as breads and breakfast cereals, in order to balance the significant increase in consumption of animal proteins and fats. Actually, the dietary guidelines have moved towards the consumption of increasing amounts of fiber during the last decade. Whole wheat flours, besides to be a fiber source provide complex carbohydrates, proteins, vitamins and minerals. However, simultaneously to the nutritional benefits, whole wheat flours contain some undesirable substances such as phytates . Phytate or myo-inositol hexaphosphate is a common constituent of most cereals, legumes, some vegetables and fruits . Phytates decrease the bioavailability of multivalent cations, due to the formation of insoluble complexes in the gastrointestinal tract. Several investigations have demonstrated that a phytate-rich diet due to a high fiber intake causes deficiencies of zinc, calcium, iron and phosphorus [3-8]. However, studies both in vivo and in vitro indicate that a partial dephosphorylation of myo-inositol hexaphosphate to lower inositol phosohates decreases the negative effect on mineral absorption [9,10]. The breadmaking process allows to decrease the phytate content. Some reports have described different breadmaking procedures aimed in lowering the phytate content in breads, for example, the phytate content in whole-meal wheat breads was half-reduced by increasing the yeast concentration [11, 12]. Conversely, Tangkonchitr et al.  reported that increasing yeast concentration did not increase phytate degradation. Knorr et al.  found that the addition of phospho-esterases (phytase, phosphatase) to the whole wheat flour, resulted in a significant reduction of phytate content in the doughs. However, all these studies followed the phytate content in doughs during the fermentation without reaching the final bread. Therefore, there is scarce information about both the phytates content and the final quality of the fresh bread.
Cereal grains contain an endogenous phytase, that catalyses the stepwise hydrolysis of phytate to phosphate and inositol via penta to monophosphates, and thereby obtain cereal products with improved mineral bio-availability [15,16]. The phytate degradation in doughs, like other enzyme reactions, depends on many factors: fermentation time, temperature, pH, water content of dough, flour extraction, yeast concentration, added mineral salts, leavening agent and breadmaking process . The aim of this study was to investigate the effect of the phytase supplementation in a breadmaking process on the technological parameters of the process and on the quality of the fresh bread. In order to get a more valid information about the effect of phytase addition in a breadmaking process, different fiber-rich bread formulations were tested. In addition the phytate content and also the phytase activity through the breadmaking process were determined and their content compared in the different formulations with the objective of select a fiber-rich formulation with the minor content of phytates.
EXPERIMENTAL PROCEDURE Materials Whole wheat flour, white wheat flour, rye flour, carob fiber and wheat bran from the commercial market were used in the preparation of the breads. Instant active dry yeast was used as starter. Four varieties of fiber-rich breads were prepared: whole wheat bread (WB), wheat bread enriched with 5 % bran (BB), wheat bread enriched with 3 % carob fiber (CB) and rye bread (RB). The characteristics of the flours used for breadmaking processes are shown in Table 1. A commercial phytase (188.8.131.52) from Aspergillus niger was gifted by Novo Nordisk (Bioindustrial, Spain). [Table 1]
Extraction of phytates The extracts of phytates were prepared following the method reported by Latta and Eskin  modified. The extraction was carried out with 2.4 % HCl (0.65 N) as this proved to be really efficient in extracting total phytate. Ten grams of flour, dough or bread were ground in a Virtis homogeniser (3 x 10 s strokes at 20000 rpm) with 50 mL of HCl solution. The homogenate was centrifuged and supernatant was filtered throughout glass wool. The clear extract was added with 20% trichloroacetic acid (TCA) in proportion 6:1; after cooling, the TCA-insoluble proteins were removed by centrifugation at 12000 rpm for 5 min. Supernatants were stored at 4 ºC for further phytate assays.
Phytate determination The phytate content was measured by using the modified Wade reagent (0.03 % FeCl3.6H2O and 0.3 % sulfosalicylic acid in distilled water) following the method reported by Latta and Eskin  adapted to a microplate reader. The pink color of the Wade reagent is due to the reaction between ferric ion and sulfosalicylic acid with an absorbance maximum at 500 nm. In the presence of phytate the iron becomes bound to the phosphate ester and unavailable to react with sulfosalicylic acid, resulting in a decrease in the peak color intensity. The phytates content was expressed as mg of phytic acid / g in dry matter. In all cases four replicates were assayed for each experimental point.
Determination of phytase activity The enzymatic extracts were prepared following the method reported by Konietzny et al.  with slight modifications. Briefly, extracts were prepared by homogenising 10 g of flour, dough or bread in 50 mL of ice-cold 100 mM sodium acetate buffer, pH 5.0, containing 5 mM 2-mercaptoethanol and 10 mM etilendiaminotetra-acetico (EDTA), using a Virtis homogeniser 5
(3 x 10 s strokes at 2000 rpm). The homogenate was centrifuged (12000 rpm, 15 min, 4 ºC) and the supernatant was filtered throughout glass wool. The clear solution was stored at 4 ºC for further enzyme activity assays. The phytase activity was measured by using a phytic acid (dodecasodium salt, Sigma) as substrate following the method reported by Kikunaga et al.  slightly modified. The incubation mixture for phytase assessment consisted of 500 µL of 0.1 M sodium acetate buffer, pH 5.0, containing 1.2 mM sodium phytate with 100 µL of the enzymatic extract. After incubating for 20 min at 50 ºC, the enzyme reaction was stopped by adding 100 µL of 20 % TCA. The mixture was cooled in ice bath during 15 min and centrifuged at 12000 rpm for 5 min. The inorganic phosphate contained in the supernatant was measured by ammonium molylibdovanadate method  adapted to a microplate reader. Blanks were run by addition of TCA solution to assay mixture prior the addition of the enzyme extract. The phytase activity was expressed as µg P / g min in dry matter. In all cases four replicates were assayed for each experimental point.
Breadmaking procedure The bread dough formula consisted of flour from WB, BB, CB or RB (100 g), dry yeast (0.97 g), salt (2.0 g), ascorbic acid (0.010 g), water up to optimum absorption. A fungal phytase was added for comparative purposes. The ingredients were mixed for 8 min. In doughs to which phytase were added, this ingredient was first suspended in the dough water. After mixing, resting (10 min), dividing (150 g), kneading and resting (10 min), doughs were mechanical shaped, proofed (up to optimum volume increment at 29 ºC, 80 % relative humidity) and baked (180 ºC-30 min, for WB; 190 ºC-22 min, for BR and 190 ºC-20 min for CF and RB). After baking, loaves were cooled for 2 h at room temperature.
After mixing and resting a sample of dough was withdrawal, which was referred hereafter as unfermented dough.
Technological evaluation The volume increment and pH of doughs were measured during proofing. Physicochemical characteristics of breads included weight, volume (seed displacement), density, width/height ratio of the central slice, moisture content and texture (texture profile analysis, TPA) were determined. TPA using a Texture Analyser TA-XT2i (Stable Micro Systems, Surrey, UK) was performed using a 25 mm diameter plunger; a 2 cm crumb slice was compressed twice up to 50%, with an interval of 50 s between compressions. The following parameters were evaluated: hardness, springiness (elasticity), cohesiveness, chewiness and resilience. In all cases four replicates were assayed for each experimental point.
RESULTS AND DISCUSSION Influence of the fungal phytase on the breadmaking process Since phytase activity is highly dependent on pH, the pHs of the doughs were measured during proofing. The pH changes were relatively small, the pH differences between unfermented and fermented doughs were approximately 0.3 pH units in all formulations. The addition of fungal phytase did not practically modify the pH values obtained throughout the proofing process (Table 2). [Table 2] The fermentation times needed to reach the optimum volume increment for the different dough formulations are also presented in Table 2. The doughs supplemented with fungal phytase required lower fermentation times than the doughs without exogenous enzyme, therefore the
fermentation step was speeded up by the addition of phytase, without affecting the pH of the doughs.
Effect of phytase addition on fresh bread quality Regarding the fresh breads, the quality parameters are shown in Table 3. A tendency to increase the specific volume of breads could be envisaged when adding fungal phytase, although no significant differences could be found. Nevertheless, a significant improvement of the bread shape (width/height ratio) was obtained regardless whole wheat bread. The effect of phytase addition on the crumb texture of the different types of breads was also analyzed. In all formulations the hardness or firmness of the bread crumbs was reduced, so softer crumbs were obtained with phytase supplementation. Other texture parameters as gumminess and chewiness where also decreased (data not showed). [Table 3] The improving effects of phytase on the breadmaking process and bread quality are rather similar to the well-known effects promoted by the addition of α-amylases (a better gas retention during proofing, lower fermentation time, improved specific bread volume, reduced crumb firmness), although no α-amylase activity was detected in the commercial phytase. Therefore the above results could be explained by considering an activation of the endogenous α-amylases, as a consequence of the increased concentration of the free calcium ions, released from the phytate-calcium complexes when phytates are hydrolyzed. The induced hydrolysis of phytates by the supplementation of fungal phytase can increase the concentration of free calcium ions necessary for the α-amylase activity. In fact, Cawley and Mitchell  suggested that the inhibition of α-amylase activity in sprouted wheatmeal should be due to binding of calcium ions by phytic acid; although later on Sharma et al.  in their studies of α-amylase activity in different cereals suggested that the inhibitory effect of the phytic acid on the α-amylase should 8
be due to its own interaction with the enzyme, rather than the complexation of calcium by the phytic acid. In any case, the final effect would be the inhibition of the α-amylase activity by phytates, and their activation would be promoted by the supplementation of phytase via the released of either calcium ions or α-amylase depending on the explanation accepted.
Evolution of phytates through the breadmaking process Phytates are considered anti-nutritional compounds due to their adverse effects on the mineral absorption. Since some fiber-rich bread formulations have been elaborated, a comparative study of the phytates content in these breads has been developed. Initially, the flours used in this study were characterized by determining the phytic acid content. Whole wheat flour had higher phytic acid content than the other flours, being the lowest content observed in the flour containing rye (Fig. 1). This can be explained by the heterogeneous distribution of the phytic acid in the wheat and rye grain. It is abundant in the germ and in the external layer (mainly in the aleurone), and scarce in the endosperm . Consequently the content of phytic acid differ considerably in white flour, whole flour and bran (4.4; 7.4 and 37.7 mg / g, respectively). Therefore the presence of bran or dietary fiber in breads leads to a high proportion of phytates. The phytic acid content of flour was correlated with the ash content, as previously reported by Fretzdorff and Weipert . [Figure 1] A decrease of the phytates was observed during the breadmaking process (Fig. 1), as a consequence of the phytase activity. Therefore the decrease of phytic acid content should be attributed to the endogenous phytase activity. In all bread formulations, the highest reduction of phytic acid was observed after mixing and resting, and only a very slow decrease was further determined. The phytate in WB dough was reduced by 28.2 % after mixing and resting, and it 9
drop to 61.3 % at the end of the fermentation; similar results were reported by Türk et al.  and Türk and Sandberg . The final content in the whole wheat bread was 4.2 mg phytic acid / g in dry matter, that is a reduction of 43.5 % regarding to the initial value in the flour. Different results have been found in the scientific literature, McKenzie-Parnell and Davies  found that bread made from whole meal flour lost 30-48 % phytate during processing and Reinhold et al.  reported that only 15-25 % of phytate was hydrolyzed in whole meal bread. Those differences could be explained by the several factors that concurred in the phytates hydrolysis (fermentation time, particle size of meals, pH, temperature, minerals content, water content, etc). However, there is no information about the phytate evolution in different formulations containing fiber, which is the main subject of this study. In Fig. 1 can also be observed the evolution of phytates in doughs enriched with bran (BB) and carob fiber (CB). The mixing and resting steps reduced the phytate content in both BB and CB doughs up to 68 % and 65 %, respectively; and a further decrease up to 77.3 % (BB) and 65.8 % (CB) trough the fermentation was observed. The residual values of phytates in BB and CB breads were 1.32 and 1.42 mg phytic acid / g in dry matter, respectively, which means a reduction of 78.3 % (BB) and 68.3 % (CB) with regard to the phytic content in the flours. Regarding the data obtained with whole wheat breads, the decrease in phytate content was significantly higher in breads formulated with flour of lower extraction rate (BB and CB) and lower ash content (Table 1), than in WB breads. Hydrolysis of phytic acid in rye dough followed a similar pattern than in BB and CB wheat doughs (Fig. 1). Phytates were reduced till to 37.3 % and 58.0 %, before and after proofing, respectively.
Effect of phytase addition on the phytates content In order to promote the hydrolysis of phytates, a fungal phytase was added to the different doughs. A higher decrease of the phytate content was obtained by adding fungal 10
phytase (Fig. 2). Hydrolysis of phytic acid in doughs added with fungal phytase followed a similar pattern to that of doughs without exogenous enzyme. The highest degradation also occurred before fermentation, but the further hydrolysis proceed at higher rate than was observed in doughs without the addition of fungal phytase. In BB and CB fermented doughs the phytic acid content decreased 89 % and 79 %, respectively; while in rye dough a decrease of 74 % was found after proofing. The baking step did not greatly affect the decomposition of phytate in these formulations, the content of phytates in the doughs and breads were similar. In the whole wheat formulation only slight differences were promoted by the addition of phytase; after resting the phytate of WB was reduced 36.2 % of the initial flour value, and drop 49.4 % after proofing. The residual values of phytates in WB bread was 3.74 mg phytic acid / g in dry matter. The above results indicate that neither the enzyme addition, the phytates were not fully hydrolyzed. In this bread formulation the total removal of phytate by adding exogenous phytase was difficult, probably due to the particle size of the bran as pointed out Sandberg and Svanberg . [Figure 2] Phytase activity through the breadmaking process The phytase activities in the tested flours are shown in Fig. 3. The phytase activities did not follow the same trend than the phytic acid content. The enzyme activity was higher in rye flour than in wheat flours, in agreement with previous findings of Harland and Harland  and Fretzdorff and Brümmer . Like the phytic acid, phytase has an heterogeneous distribution in the cereal grain, being the activity greater in the aleurone layer . Throughout the breadmaking process, diverse results were obtained depending on the type of bread considered. In the rye bread processing, the phytase activity showed a progressive decrease, while in the BB and CB an important decrease of the phytase activity was observed in the first step, namely after mixing and resting. No noticeable change in the enzyme activity was detected in the whole 11
wheat bread processing. These results could only be explained by the inhibition promoted by the reaction products, since no denaturation or deactivation of the enzyme could be occurred during the fermentation process. It is well-known the inhibition of phytase provoked by the phosphorous resulting from the hydrolysis of phytates  nevertheless, previous differences in enzyme activity thorough the breadmaking can be attributed to differences in the phosphorous concentration present in the doughs. Apparently the phytase are still active during the early stages of the baking process, but in breads no residual activity could be detected; it was concluded that cereal phytases were inactivated during baking. [Figure 3] When fungal phytase were supplemented, the phytase activities ranged from 147.0 (BB) to 218.5 µg P / g min (RB), and the activity was hardly affected throughout the breadmaking process (data not showed). The fungal phytase also was inactivated during baking; no phytase activity was detected in fresh breads.
CONCLUSIONS From the above results, it can be concluded that phytase supplementation as a breadmaking additive in bread formulations containing fiber leads to an acceleration of the proofing, an improvement of the bread shape, an slight increase of the specific volume and also confers softness to the crumb. In addition, from the nutritional point of view a further hydrolysis of the phytates (considered as anti-nutritional compounds) is reached by adding exogenous phytase, so an improvement in the mineral adsorption can be obtained with the consumption of phytase supplemented breads. Therefore, the phytase presents adequate properties to be used as breadmaking additive.
Regarding the four formulations tested, it could be pointed out that the one with carob fiber could be the recommended one because of both its fiber content and phytate content, and the quality of the fresh bread quality originated.
Acknowledgements This work was financially supported by European Union and Comisión Interministerial de Ciencia y Tecnología Project (FEDER, IFD97-0671-C02-01) and Consejo Superior de Investigaciones Científicas (CSIC), Spain. M. Haros would like to thank the post-doctoral grant from program René Hugo Thalmann of the Universidad de Buenos Aires, Argentine.
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Table 1 Characteristics of the flour used WB
Protein (%, d.m.)
Ash (%, d.m.)
Water Absorption (%)
WB: Whole wheat flour, 100% extraction rate BB: white wheat flour + wheat bran (5%) CB: white wheat flour + carob fiber (3%) RB: rye flour (51%) + white wheat flour (49%)
Table 2 Effect of adding fungal phytase on dough behavior during proofing Dough
Proofing time (min)
whole wheat flour (WB), white wheat flour + wheat bran (BB), white wheat flour + carob fiber (CB) and rye flour + white wheat flour (RB) (-) without fungal phytase (+) with fungal phytase
Table 3 Effect of adding fungal phytase on technological parameters of fresh bread Moisture
36.9 ± 0.1
2.71 ± 0.08
1.48 ± 0.03
1470 ± 18
36.3 ± 0.1
2.72 ± 0.02
1.35 ± 0.13
1405 ± 42
33.1 ± 0.1
3.54 ± 0.06
1.31 ± 0.01
853 ± 64
31.8 ± 0.1
3.62 ± 0.04
1.16 ± 0.03
699 ± 70
31.8 ± 0.1
3.51 ± 0.09
1.24 ± 0.07
755 ± 51
32.3 ± 0.1
3.75 ± 0.06
1.12 ± 0.03
654 ± 39
34.2 ± 0.1
2.48 ± 0.09
1.41 ± 0.03
1453 ± 95
34.8 ± 0.1
2.52 ± 0.09
1.33 ± 0.01
1251 ± 86
Whole wheat flour (WB), white wheat flour + wheat bran (BB), white wheat flour + carob fiber (CB) and rye flour + white wheat flour (RB) (-) without fungal phytase (+) with fungal phytase Mean ± standard deviation (four replicates were run)
Fig. 1 Phytic acid contents in flour, doughs and breads at different stages of the breadmaking process. Whole wheat flour (WB), white wheat flour + wheat bran (BB), white wheat flour + carob fiber (CB) and rye flour + white wheat flour (RB).
Fig. 2 Effects of adding fungal phytase on phytate degradation in different doughs and breads formulations during breadmaking process. Whole wheat flour (WB), white wheat flour + wheat bran (BB), white wheat flour + carob fiber (CB) and rye flour + white wheat flour (RB).
Fig. 3 Phytase activities in flour, doughs and breads at different stages of the breadmaking process. Whole wheat flour (WB), white wheat flour + wheat bran (BB), white wheat flour + carob fiber (CB) and rye flour + white wheat flour (RB).
Phytic Acid Content (mg / g)
Phytic Acid Content (mg / g)
Phytase Activity (µg P / g min)
RB WB CB
BB fermented dough