FUNGAL PHYTASES IN WHOLEMEAL BREADMAKING I: 3-PHYTASE A IMPROVES STORAGE STABILITY AND IN VITRO DIGESTIBILITY OF NUTRIENTS IN WHEAT BREADS

Krzysztof Żyła¹, Magdalena Mika¹, Halina Gambuś², Anna Nowotny², Beata Szymczyk^3
1 Department of Food Biotechnology, University of Agriculture in Cracow, Poland
² Department of Carbohydrates Technology, Agricultural University of Cracow, Poland
³ National Research Institute of Animal Production Department of Animal Nutrition and Feed Science in Aleksandrowice, Poland

ABSTRACT

Microbial 3-phytase A at seven activity levels ranging from 0 to 5000 FTU/kg was tested as a breadmaking additive in baking of wholemeal wheat breads. No consistent relationships between levels of 3-phytase A and bread weights, bread volumes, baking yield or total baking loss were found. There were no effects of the enzyme addition on bread moisture. The enzyme significantly improved firmness (p<0.001) and chewiness (p<0.001) of fresh breads in a dose-depended manner. The improvements in texture over control breads were preserved during 3 days of storage. 3-Phytase A at 5000 FTU/kg increased bread volume by 11 % and in vitro digestibilities of phosphorus, calcium, carbohydrates, and protein by 45, 16, 11, and 56%, respectively. In control breads, phytate content of the wholemeal wheat flour was reduced by 51%. The extend of the subsequent loss of phytate in breads supplemented with 3-phytase A was reciprocally related to the enzyme activity applied (R²= 0.975; p<0.001) and reached 92% in breads supplemented with 5000 FTU/kg. Myo-inositol pentaphosphates (IP5) concentrations in control breads and in breads baked with 3-phytase A at 30-1000 FTU/kg were 5 fold higher than in flour whereas in breads baked with the enzyme at 5000 FTU/kg no IP5 were found.

Key words: wheat, bread, phytate, 3-phytase A, storage stability, nutrient digestibility.

INTRODUCTION

Foods based on cereal grain contain large proportions of functional substances, including minerals, trace elements (magnesium, zinc and manganese), antioxidants, vitamins, fermentable carbohydrates (e.g. inulin, resistant starch, oligosaccharides), phytoestrogens, and antinutrients. Increased intake of the whole-grain foods has been related to reduced risk of developing type 2 diabetes, cardiovascular disease, stroke and hormonally depended cancers [6, 17]. Apart from all these generally known nutritionally active factors, foods based on cereals are also high in phytate (IP6) – salts of phytic acid (myo-inositol hexakisdihydrogenphosphate). Phytate phosphorus comprises about two-thirds of the total phosphorus content in cereal grains and the presence of phytate in foods relates to reduced bioavailability of calcium, magnesium, zinc, iron, and protein [12]. Phytate, as well as different myo-inositol phosphate esters, products of enzymatic hydrolysis of phytate, vary in their inhibitory effect on pepsin, α-amylases, and show different chelating capacity toward nutrients. Physiological pH values alter solubility of phytate complexes with minerals and protein. As a result, digestibility of proteins, starch and lipids is reduced [21]. On the other hand, however, it has been demonstrated that sodium phytate solutions may have striking anticancer effects (preventive and therapeutic) both in vitro and in vivo [24].

Phytases are enzymes that hydrolyse phytate usually to myo-inositol-2-monophosphate and inorganic phosphorus in a stepwise manner forming myo-inositol phosphate intermediates (IP5, IP4, IP3, IP2) [10, 29]. The action of plant or microbial phytases on phytate deprives this component of antinutritional properties when dephosphorylation renders myo-inositol esters with less than five phosphate moieties [23]. Commercial availability of Aspergillus niger (ficuum) 3-phytase A (EC 3.1.3.8) for more than two decades has resulted in numerous studies on properties and ability of the enzyme to increase digestibility and bioavailability of minerals and protein in both animals and humans.

It is a well known phenomenon that during breadmaking process phytate content of the dough is considerably reduced. The effect has been found to depend on different factors like flour extraction rate, proofing time and temperature, acidity of the dough, the presence of calcium, and has been attributed to the catalytic activity of endogenous cereal phytases rather than to the activity of the yeast phytase [27]. The low optimum pH (3.5) as well as the repression of the enzyme expression by both high pH and high inorganic phosphorus concentrations encountered at typical leavening conditions, limit contribution of yeast phytase to hydrolysis of phytate during breadmaking [1]. Certain modifications of baking technology, like soaking and malting or the sour dough lactic acid fermentation, have been found to stimulate phytate degradation during breadmaking [14, 20]. Türk & Sandberg [26] were the first to study effects of the addition of microbial 3-phytase A to wheat dough on phytate hydrolysis during breadmaking. More recently, the efficacy of the enzyme as a potential breadmaking additive has been studied by Haros and co-workers [4, 5]. In those studies a significant reduction of phytate content in wheat breads supplemented with phytase, in addition to increased bread volume and improved crumb texture have been documented. All the effects however, have been attained at relatively high levels of a microbial phytase that creates concerns about limited practical application of enzyme technology, especially in comparison to genetically modified plants with reduced phytate contents [15]. An improvement in iron dialyzability in wheat breads made with commercial phytases has been documented [19] but scarce information is available about changes in bioavailability of nutrients that accompany phytate hydrolysis by exogenous microbial phytases. Although improvements of crumb texture in enzymatically modified breads have been studied thoroughly, nothing is known about possible antistaling effect of phytase.

The purpose of the current studies was to learn effects of different levels of a commercial preparation of fungal 3-phytase A on breadmaking performance, crumb texture parameters and on changes in crumb softness during 3 days of storage. Yet another objective was to study the influence of the enzyme on phytate hydrolysis and nutrients release from wholemeal wheat breads in an in vitro model that simulated digestion in humans.

MATERIALS AND METHODS

Materials

Wholemeal wheat flour (100 % extraction rate) was from a local flourmill (Kraków, Poland). “Dry acid” – a mixture of organic acids (lactic, citric, and acetic acids) used in bakeries, and compressed yeasts were obtained from Lesaffre P.S. (Łódz, Poland).

Enzyme

“Finase P” – a commercial preparation of microbial 3-phytase A (EC 3.1.3.8) used in this study had the phytase activity of 5.250 units/g (declared by the producer; AB Enzymes GmbH, Darmstadt, Germany). The enzyme is synthesized by a genetically modified Trichoderma reesei GMO strain carrying the phyA gene.

Enzyme Activity Measurements and Units

The activity of phytase A was determined using the procedure described previously [30]. One unit of phytase activity (FTU) was defined as the amount of enzyme required to liberate 1 μmol inorganic phosphorus from 5.5 mmol sodium phytate in 1 minute at 40°C, pH 5.0. The measured activity of phytase in “Finase P” was 4.132 FTU/g. In making of wheat breads, 3-phytase A was added at 0, 30, 60, 240, 1000, 2000, and 5000 FTU/kg of flour.

Breadmaking procedure

The dough formulation for wheat breads was the following: wholemeal wheat flour (1000 g), compressed yeast (30 g), salt (20 g), dry acid (10 g) and water (670 mL). Aliquots of phytase preparation were dissolved in a small part of water and mixed with its remaining part. Baking ingredients were mixed for 9 minutes, rested for 10 minutes, divided (250 g), put into forms, proofed for 150 minutes at 35°C and baked (230°C, 30 min) in a laboratory oven.

Technological evaluation

During proofing, pH of doughs was determined potentiometrically. Baking yield and total baking loss were assessed by standard procedures. In breads, weight, volume, moisture content and texture profile were evaluated. Texture profile analysis was performed on the TX-XTA texture analyser (Stable Micro Systems, Surrey, UK). The analysis comprised firmness, springiness, cohesiveness, chewiness and resilience determinations. Storage stability was determined in breads wrapped in commercial plastic bags and kept in chambers at 24^oC and relative humidity of 64% for 3 days after baking. Moisture content and texture profile of experimental breads were determined after 24, 48 and 72 hours of storage.

Determination of myo-inositol phosphates

Samples of powdered breads were defatted, mixed with 0.4M HClO₄ and extracted with a shaker for 2 h. The extract was centrifuged, neutralized with 1M K₂CO₃ and put on a HyperSep NH2 SPE column. A fraction eluted from the column by 0.6 mL 1M HCl was used for HPLC analysis of inositol phosphates. Inositol phosphates (IP6-4) were determined by a modified Lehrfeld procedure [11] using ion-pair C18 reverse phase HPLC with refractive index detection.

In vitro determination of nutrients digestibility

An in vitro procedure designed to simulate digestion in the human intestinal tract was used. Triplicate samples (0.4 ą 0.001 g) of bread powders were weighted into plastic syringes without Luer-lock tips. Samples were hydrated with 1.0 mL of water and an HCl solution so that a pH value of 2.0 was obtained. Then 6200 units of pepsin were added, and the contents of each tube were mixed, sealed with parafilm, vortexed, and incubated in a water bath at 37°C for 2 hours. At the end of this period, 0.5 mL of a NaHCO₃ solution along with 2 mg of pancreatin and 25 mg of bile extract (0.6 mL) were added to reach pH 7.0. The slurry was transferred quantitatively to segments of dialysis tubings by means of syringe piston. Segments were placed in 250 mL Erlenmeyer flasks containing 40 mL of 0.1 M NaCl in 0.05M imidazole buffer pH 7.0 and were incubated in a shaking water bath. After 4 hours samples of the dialysate were withdrawn for determination of inorganic phosphate, calcium, protein and reducing sugars as detailed elsewhere [31].

Statistical analyses

Data were subjected to one-way or two-way analysis of variance using Statgraphics Plus for Windows 5.1 statistical package. Mean differences were determined using Fisher’s least significant difference test. Statistical significance was accepted at p <0.05.

RESULTS

Although there were significant effects of different phytase levels added to wholemeal wheat dough on bread weights, bread volumes, baking yield and total baking loss (Table 1), no strict relationships between phytase activity and the baking parameters were found.

There were no effects of phytase addition on the moisture of enzymatically modified wheat breads. Breads baked with exogenous phytase at 30 FTU/kg had significantly lower weights, lower baking yields and a significantly higher total baking loss than the control breads prepared without phytase addition. Similar effect of decreased weights was found in breads supplemented with phytase at 240 FTU/kg. In breads baked with the highest phytase activity tested (5000 FTU/kg) loaf volumes were increased by 11 %.

Both the level of phytase activity and the time of storage significantly affected firmness and chewiness of the wholemeal wheat breads (Table 2).

The springiness, cohesiveness, and resilience of breads, however, were not affected by the enzyme addition (data not shown). The overall values of firmness and chewiness, calculated for all the phytase activity levels tested, in breads stored in chambers with controlled constant temperature and humidity for three days, increased linearly with the time of storage and, on completion of the experiment exceeded their initial values by 90 and 22 %, respectively. Generally, the firmness and chewiness were significantly improved at certain low levels of the activity of 3-phytase A, but the most pronounced, linear effects were observed when phytase activity added during dough preparation exceeded 1000 FTU/kg. On the other hand, however, even the lowest activity level of 3-phytase A tested in the experiment (30 FTU/kg) significantly reduced values of both firmness and chewiness of experimental breads. The increase in microbial phytase addition from 30 to 240 FTU per kg of flour did not change the mean values of firmness and chewiness calculated for the entire three days period of bread storage. There were also significant interactions found between phytase dosage and the storage time for both firmness and chewiness of breads. The nature of these interactions was by no means straightforward but it seems that as the storage time increased, higher activities of phytase were necessary for significant improvements in the firmness and chewiness of breads. It should be emphasized that after three days of storage the firmness of wheat breads baked with the phytase activity at 5000 FTU/kg was 2.23 kG that is comparable to 2.28 kG, the firmness of fresh breads made without enzyme addition. After three days of storage the improvement in chewiness resulting from phytase addition at 5000 FTU/kg was even more pronounced (0.70 kG for breads made with 3-phytase A vs. 0.97 kG for the fresh control breads).

As the activities of 3-phytase A added to wheat doughs were increased significant increases in amounts of phosphorus, calcium, reducing sugars and peptides (α-amino nitrogen) released from bread powders by the in vitro procedure of multiple digestions were observed (Table 3).

Amounts of dialyzable phosphorus were increased by almost each increment in phytase dosage, and, at 5000 FTU/kg, exceeded dialyzable phosphorus content in control breads by more than 30 %. As compared to the control breads, the concentration of α-amino nitrogen released from bread samples was significantly enhanced at phytase dosage of 240 FTU/kg or higher, whereas for altering the concentrations of calcium and dialyzable sugars, phytase effective dosage was 1000, and 2000 FTU/kg, respectively. Amounts of calcium freed from experimental breads were additionally enhanced in breads supplemented with 3-phytase A at 5000 FTU/kg. Less than 50% of phytate (IP6) concentration in the wholemeal wheat flour was found in control breads made without phytase addition. The addition of exogenous microbial 3-phytase A during dough preparation promoted phytate degradation in a dose depended manner and at 5000 FTU/kg only 8.4 % of its original content in flour was detected in experimental breads. A meaningful reciprocal relationship [IP6 = 1/ (3.45 + 0.0029 x phytase); R²= 0.98; p<0.001] was calculated between the activity of 3-phytase A added to the dough and concentrations of IP6 in experimental breads. Amounts of myo-inositol pentaphosphates (IP5) in control breads and in breads supplemented with 3-phytase A at less than 2000 FTU/kg were 3-4 fold higher than in the wholemeal wheat flour. At the phytase supplementation level of 2000 FTU/kg, however, the amounts of IP5 were substantially reduced, and at 5000 FTU/kg no IP5 were detected in experimental breads.

DISCUSSION

3-phytase A as a possible breadmaking improver has already been tested with the aim to accelerate phytate hydrolysis in the dough and to decrease phytate content in wheat bread [19, 26]. Apart from possible nutritional benefits related to reduced phytate levels, Haros with co-workers [4, 5] have documented important technological advantages of high phytase levels added to wheat doughs, like a shortened fermentation time, increased specific loaf volume, and improved crumb texture. Our observations and experimental data presented here confirmed technological benefits resulting from high dosages of phytase application. In breads baked with 3-phytase A at 5000 FTU/kg significant enhancements in bread volumes were observed. During baking, starch gelatinisation is thought to be responsible for major structural changes that include interactions of partially solubilised starch granules with protein and fat fraction of wheat [2]. Contradictory data about the contribution of different wheat constituents to loaf volume are provided by the literature. Matsuoukas & Morrison [16] found the ratio non-polar/polar free lipids in wheat to be highly correlated with loaf volumes while McCormack with co-workers [18] claimed that in comparison to protein, lipid contribution to loaf volumes was negligible. However, the beneficial effects of lipase added to dough or secreted by genetically engineered yeasts on loaf volume [8, 22] support the current consensus that thermostable amylose-lipid complexes on one hand, and occlusion of lipids within the gluten network on the other, are of utmost importance for a proper bread volume [3]. In the study presented here, similarly to the study of Haros et al. [4], 3-phytase A might enhance starch amylolysis by releasing additional amounts of calcium from calcium-phytate complexes that in turn activated endogenous alpha-amylase of wheat. Bread volumes depended on carbohydrate degradation much stronger than on the breakdown of calcium-phytate complexes since there was a closer relationship between amounts of reducing sugars freed breads in vitro and the volumes of experimental breads (R² = 0.54; p < 0.0001), than between bread volumes and calcium release (R² = 0.39; p < 0.0025). There might have been additional factors beside phytate hydrolysis, therefore, that contributed to carbohydrate degradation in breads. The detrimental effect of phytase on bread weights observed at certain low activities of the enzyme is hard to explain. It seems possible that at lower activities of phytase, free calcium concentration was insufficient for alpha-amylase activation and certain wheat constituents might bind this element.

Different factors have been credited for texture improving properties during breadmaking and the mechanism behind their action is usually explained by extended amylolysis of starch that produces short-chain maltodextrins. Jimenez and Martines-Anaya [7] reported that while low molecular weight dextrins generated from starch by alpha-amylase correlated with fresh bread properties and did not induce changes in texture during storage, water-insoluble pentosans correlated with crumb elasticity and hardness. In our studies, even the lowest phytase activity tested (30 FTU/kg) caused significant improvements in the texture of wheat breads reducing considerably crumb firmness and chewiness. The enhanced crumb softness cannot be attributed to increased alpha-amylase activity because high phytase activity (1000 FTU/kg) was needed for a significant increase in the in vitro digestibility of calcium. Furthermore, the improvements in crumb texture resulting from the application of 3-phytase A were preserved for at least 3 days of storage and the effect was positively related to levels of phytase activity applied. Although different enzyme activities have been proven to exert antistaling effect in baking [13, 25] no form of phytase has ever been mentioned in this context. The preparation of 3-phytase A employed in current studies is synthesized by a genetically engineered Trichoderma reesei strain carrying the phyA gene and is high in side activities acquired from the host genome. Using the data and definitions of activity units provided by Wikiera [28] it may calculated that 30 FTU in the “Finase P” preparation was accompanied by 3.3, 0.96, 2.1, 1.29, 2.16 units of poligalacturonase, acid proteinase, xylanase, β-glucanase, and cellulase activity, respectively. It seems possible that especially at low levels of the enzyme supplementation Trichoderma side activities might contribute to the improvements in texture more than the activation of endogenous wheat alpha–amylase by calcium released from complexes with phytate. The side xylanase activity from Trichoderma might also be the additional factor that contributed to improved volume of breads.

Similarly to observations reported before [4,5,9,26], a considerable hydrolysis (51%) of phytates in dough by endogenous plant phytases with subsequent increases in the concentration of myo-inositol pentaphosphates was observed in this study. In the literature, the extent of phytate hydrolysis by endogenous phytate-degrading enzymes of wheat flour varying from 24 [4] to 96% [27] has been reported. These differences may be attributed to varying levels of endogenous phytase activity of wheat, different dough formulations and also to differences in the process conditions. In our study, increasing activities of microbial 3-phytase A caused significant decreases in phytate concentration and with the highest dosage of the enzyme, more than 90% of IP6 and 100% of IP5 were converted into lower phosphate esters of myo-inositol. Similar level of phytate dephosphorylation (85%) in breads supplemented with a fungal phytase has been reported by Porres et al.[19]. Knorr with co-workers [9], while fortifying wheat doughs with 2 % of exogenous preparations of wheat phytase and phosphatase have reduced phytate concentration in breads by 88 %. Haros et al. [4, 5] also using a very high dosage of microbial phytase have achieved 60% of phytate dephosphorylation whereas Türk and Sandberg [26] reported complete dephosphorylation of phytates in wheat breads under similar conditions. Phytate dephosphorylation in breads supplemented with different activities of microbial 3-phytase A was, in the current study, accompanied by significant increases in amounts of inorganic phosphorus, reducing sugars and alpha-amino nitrogen released from breads by the in vitro digestions. Significantly enhanced in vitro digestibility of protein was attained with phytase activity of at least 240 FTU/kg, but no less than 2000 FTU/kg was necessary for improvements in the digestibility of carbohydrates. The best breadmaking performance parameters, the highest improvements in texture, the highest level of dephosphorylation and the highest in vitro digestibility of nutrients were attained with microbial 3-phytase A added to dough at 5000 FTU/kg. The effective enzyme dosage was ten fold higher than phytase activity usually applied for supplementation of commercial feeds fed to monogastrics [12]. At the time being, the practical application of 3-phytase A in wheat breadmaking seems therefore, be limited to special purposes.

CONCLUSION

1. In wheat breadmaking, fungal 3-phytase A at inclusion levels ranging from 2000 – 5000 FTU/kg, increases loaf volume, improves crumb firmness, exerts positive effects on in vitro digestibility of nutrients and completely hydrolyze myo-inositol hexa- and pentaphosphates.

2. Fungal 3-phytase A may be used as an antistaling agent. The improvements in crumb firmness and chewiness depend on the enzyme activity applied and are retained during 3 days of bread storage.

3. Side Trichoderma activities that accompany 3-phytase A in the “Finase P” preparation contribute probably to the efficacy of the enzyme in breadmaking.
   

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   Krzysztof Żyła
   Department of Food Biotechnology,
   University of Agriculture in Cracow, Poland
   Balicka 122, 30-149 Cracow, Poland
   phone/fax: (+4812) 662 4794
   email: kzyla@ar.krakow.pl
   
   Magdalena Mika
   Department of Food Biotechnology,
   University of Agriculture in Cracow, Poland
   Balicka 122, 30-149 Cracow, Poland
   
   Halina Gambuś
   Department of Carbohydrates Technology,
   Agricultural University of Cracow, Poland
   Balicka 122, 30-149 Cracow, Poland
   
   Anna Nowotny
   Department of Carbohydrates Technology,
   Agricultural University of Cracow, Poland
   Balicka 122, 30-149 Cracow, Poland
   
   Beata Szymczyk
   National Research Institute of Animal Production
   Department of Animal Nutrition and Feed Science in Aleksandrowice, Poland
   32-083 Balice, Poland
   

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