FUNGAL PHYTASES IN WHOLEMEAL BREADMAKING II: EFFECTS OF DIFFERENT PHYTASES ON TECHNOLOGICAL AND FUNCTIONAL PARAMETERS OF RYE 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- and 6-phytases A at 5000 FTU/kg, and their combinations with phytase B at 30 AcPU/g, were tested as breadmaking improvers in baking of wholemeal rye breads. Both phytases A increased bread volumes and the subsequent addition of phytase B amplified the effect. 6-Phytase A alone, and, especially in combination with phytase B, significantly reduced crumb firmness and chewiness. The improvements in texture were preserved during 3 days of storage. 6-Phytase A, but not 3-phytase A, reduced fat content of breads by more than 50 %, and slightly decreased protein concentration. In breads supplemented with 0.2% of myo-inositol, similar, although less pronounced, changes in fat and protein content were observed.

Key words: rye bread, 3- and 6-phytase A, phytase B, texture, antistaling.

INTRODUCTION

Preparations of several enzymes of microbial origin like amylases, proteases, xylanases and lipases have already been tested as breadmaking improvers. Although different enzymes have been proven to exert antistaling effect in baking [11, 15], no form of phytase has ever been mentioned in this context. Until now, the application of exogenous phytases as a possible breadmaking improver has been tested with the aim to accelerate phytate hydrolysis in the dough and to decrease phytate content in wheat bread [17, 14]. Apart from possible nutritional benefits connected with reduced phytate levels, Haros with co-workers [8, 9] 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. In the previous part of the study on phytases in baking [20] we confirmed that in wholemeal wheat breads supplemented with 3-phytase A at 5000 FTU/kg, loaf volumes were significantly increased, firmness and chewiness were substantially reduced, and the improvements in texture were preserved during 3 days of storage.

In rye breadmaking phytate is usually hydrolyzed to a much higher extent than in the processing of wheat breads mainly because of higher endogenous activities of phytate-degrading enzymes in grain, a significant contribution of phytate-degrading enzymes from lactic acid bacteria in the sour dough process, and lower pH values that promote dephosphorylation [7]. This explains why the application of exogenous phytases in rye breadmaking has never been tested. If one perceives, however, the phytate molecule not only as a chelating agent whose antinutritional properties should be reduced but also as a source of myo-inositol and lower phosphates of myo-inositol that may exert specific physiological effects [21], different phytases are the tools of choice for producing bread with functional properties.

In contrast to 3-phytase A, that hydrolyses first D-3 phosphate residue on the myo-inositol ring of phytate, 6-phytase A (EC 3.1.3.26) removes first the L-6 residue. Such enzyme, although found mainly in plant seeds, has been commercially available as microbial (Peniophora lycii) 6-phytase A engineered in an Aspergillus oryzae host, for a couple of years [18]. Phytase B, the enzyme with broad substrate specificity, more commonly known as a non specific acid phosphomonoesterase (EC 3.1.3.2), that removes phosphate groups from many different compounds and dephosphorylates also myo-inositol phosphates lower than hexaphosphate is also available, at least in experimental preparations.

The objective of the current study was to evaluate effects of different types of commercially available microbial phytase preparations in baking technology of wholemeal rye breads on technological parameters and on the texture of breads as well as to learn effects of enzymatic modifications on their chemical composition.

MATERIALS AND METHODS

Materials

Wholemeal rye flour was from a local flourmill (Kraków, Poland). “Dry acid” – a mixture of organic acids (lactic, citric, and acetic acids) used in bakeries, and dry instant yeast was obtained from Lesaffre P.S. (ŁódŸ, Poland). Myo-inositol was purchased from Sigma Chemicals Inc.

Enzymes

A commercial preparation of microbial 3-phytase A (EC 3.1.3.8) used in this study had phytase activity of 5.250 units/g (declared by the producer, AB Enzymes OY, Finland). The enzyme is synthesized by a genetically engineered Trichoderma reesei strain carrying the phyA gene. A preparation of 6-phytase A (EC 3.1.3.26) had the declared activity of 2.583 units/g and is produced by a genetically modified Aspergillus oryzae strain that hosts phyA gene from the fungus Peniophora lycci (Basidiomycetes class). A preparation of phytase B (acid phosphatase; EC 3.1.3.2) was obtained from the same supplier as in the previous study [21] and had acid phosphatase declared activity of 184 000 units/g.

Enzyme activity measurements and units

Activities of phytases A and acid phosphatase were determined using procedures described previously, and units of enzymic activity used in the current study were also the same as in the previous work [19]. Measured activities of the preparations were: 4.132 FTU/g, 3.221 FTU/g, and 12.680 AcPU/g for 3-phytase A, 6-phytase A, and phytase B, respectively. During preparation of rye breads 3- and 6-phytase A were added at 5,000 FTU/kg, whereas phytase B was used at 30 AcPU/g.

Breadmaking procedure

The dough formulation for rye breads was the following: wholemeal rye flour (1,000g), dry acid (80g), yeast (50 g), salt (30 g) and water (675 mL). Phytases were dissolved in a small part of water and and mixed with its remaining part. Myo-inositol was added at 2 g/kg. 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, pHs of doughs were 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) and comprised firmness, springiness, cohesiveness, chewiness and resilience determinations. Storage stability of breads was analyzed in the course of 3 days after baking. Breads wrapped in commercial plastic bags were kept in chambers at 24°C and 64% relative humidity. Moisture content and texture profile were determined after 24, 48 and 72 hours of storage. Proximate analyses of breads were performed using a Tecator auto-analyzer.

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

Effects of two forms of phytase A and of phytase B on breadmaking performance of rye breads and on the wholemeal rye breads storage stability

In making of the wholemeal rye breads, two different forms of phytase A (3- or 6-phytase at 5000 FTU/kg) alone, as well as in combination with phytase B (at 30 AcPU/g) did not affect bread weights, baking yield, or total baking loss. There were however significant effects of enzymes addition on loaf volumes and moisture contents in breads (Table 1). Bread volumes were increased by more than 6 % due to addition of either of phytases A to the dough and the simultaneous addition of a phytase A and phytase B enhanced the parameter by more than twenty percent. As compared to control breads or breads made with the addition of single phytase A, rye breads made with the combination of phytases A and B had significantly reduced moisture content

Table 1. The influence of different forms of phytases on the breadmaking performance parameters of wholemeal rye breads

Phytase added	Bread weight	Bread volume	Baking yield	Total baking loss	Moisture
	(g)	(mL)	(%)	(%)	(%)
None	216	358a	145.4	13.2	45.81b
3-A	220	390b	147.5	12.0	45.98b
6-A	219	381b	145.1	13.4	45.82b
3-A +B	217	432c	147.0	12.3	44.98a
6-A +B	216	444c	144.4	13.8	45.20a
ANOVA
SEM	2.45	4.572	1.616	0.765	0.149
p	0.909	0.0081	0.7751	0.6211	0.0492

3- or 6-phytase A added to dough at 5.000 FTU/ kg. phytase B at 30 AcPU/g a-c – Means within columns with no common superscript letter differ significantly at p<0.05

Among different texture parameters tested in the study, only the firmness and chewiness of rye breads were affected by the type of enzyme and the time of storage (Table 2). There was a clear linear relationship between time of storage and the firmness and chewiness of breads. In the course of storage that lasted three days, the firmness of breads increased by 61 % while the chewiness by 42 %. 6-Phytase A alone, and especially in combination with phytase B, was particularly effective in conserving texture parameters of breads. After three days of storage, rye breads made with 6-phytase A and phytase B had lower firmness (2.20 kG) than fresh breads prepared without enzyme addition (2.32 kG). The chewiness of fresh breads made with the combination of 6-phytase A and phytase B was more than two fold lower than in control breads and after three days of storage was still 62 % lower than chewiness of the fresh control breads. 3-Phytase A had in fact detrimental effect on the firmness but it clearly improved chewiness of rye breads.

Table 2. Effects of different forms of phytases and of storage time on the firmness and chewiness of the wholemeal rye breads

Phytase	Days of storage
	Firmness (kG)					Chewiness (kG)
	0	1	2	3	mean	0	1	2	3	mean
None	2.33	2.93	3.45	3.60	3.07d	0.99	1.04	1.14	1.57	1.19d
3A	2.71	3.14	3.58	3.84	3.31e	0.85	0.96	1.05	1.17	1.01c
6A	1.74	2.58	2.72	3.30	2.58b	0.67	0.85	0.85	0.96	0.83b
3A+B	2.12	2.83	3.01	3.46	2.85c	0.72	0.84	0.83	0.92	0.82b
6A+B	1.33	1.79	1.85	2.20	1.79a	0.46	0.51	0.57	0.61	0.54a
mean	2.04A	2.65B	2.92C	3.28D	2.72	0.74A	0.84B	0.89C	1.05D	0.88
SEM	0.130					0.030
ANOVA
phytase	0.001					0.001
time	0.001					0.001
Interaction	0.400					0.001

3- or 6-phytase A added to dough at 5000 FTU/kg. phytase B at 30 AcPU/g Two-way ANOVA on treatments. Treatment means within rows and columns with no common superscript letter differ significantly at p<0.05

Effects of two forms of phytase A alone and in combination with phytase B and of myo-inositol on chemical composition of rye breads

Rye breads prepared with the addition of phytases A and B were supposed to have negligible contents of phytate and increased concentrations of free myo-inositol. Additional breads supplemented with 0.2 % of myo-inositol were prepared therefore to learn possible effects of this compound on bread chemical composition. The breadmaking additives (enzymes and myo-inositol) significantly influenced the ash, protein and fat contents but had no effect on the fiber, carbohydrates, and total phosphorus and calcium concentrations in experimental breads (Table 3). Breads prepared with the addition of 3-phytase A had a composition similar to control breads, with the exception of fat which concentration was significantly higher. The addition of phytase B along with 3-phytase A significantly decreased protein and fat content. The influence of 6-phytase A on rye breads chemical composition differed substantially from the effects exerted by 3-phytase A. In comparison to control breads made without enzymes or with myo-inositol, those prepared with the addition of 6-phytase A had higher levels of ash, lower concentrations of protein and fat concentrations reduced by more than fifty percent. The addition of phytase B on top of 6-phytase A reduced ash and protein concentration in breads whereas phytase B applied with 3-phytase A did not change the content of ash but reduced fat concentration by more than thirty percent. The pure myo-inositol caused 22 % reduction in the fat content, significantly decreased protein content but did not affect ash concentration in rye breads.

Table 3. Proximate analyses of wholemeal rye breads made with different forms of phytases or with myo-inositol

Item	Bread with added ingredient							ANOVA SEM p
	none	3-A	6-A	3A+B		6A+B	inositol
Ash (%)	4.13bc	4.14bc	4.19d		4.16cd	4.10a	4.12ab	0.0096	0.0047
Protein (%)	13.2e	13.3e	12.9c		13.0d	12.3a	12.8b	0.0245	0.0001
Fiber (%)	2.38	2.04	2.08		2.22	1.96	2.22	0.0845	0.0920
Fat (%)	1.49d	1.58e	0.68a		1.01b	0.64a	1.16c	0.0186	0.0001
Carbohydrates (%)	73.6	73.9	74.4		74.4	76.1	76.1	3.285	0.9937
P total (mg/g)	2.905	2.891	2.940		2.947	2.907	2.933	0.057	0.0984
Ca (mg/g)	0.545	0.521	0.510		0.497	0.494	0.550	0.071	0.2689

3- or 6-phytase A added to dough at 5.000 FTU kg-1 . phytase B at 30.000 AcPU kg-1. myo-inositol at 2 g/kg a- e- Means within rows with no common superscript letter differ significantly at p<0.05

DISCUSSION

In this part of the study two forms of phytase A, and namely 3-phytase A and 6-phytase A, that differ in the mode of action against phytate and have distinct affinity towards different myo-inositol phosphates, were applied alone, and in combination with a non-specific acid phosphomonoesterase (phytase B) to doughs based on wholemeal rye flour. To our knowledge, microbial phytases have never been tested in baking technology of rye breads and 6-phytase A and phytase B have never been exploited for the possibility to produce bread with unique nutritional and functional properties. Similarly to wheat breads [20], rye breads produced with the addition of 3- or 6-phytase A had enhanced volumes and the addition of phytase B on top of phytases A resulted in additional increases of the parameter. Rye breads made with exogenous phytate-degrading enzymes had different texture and chemical composition. The most striking differences between breads prepared with 3-phytase A and those made with 6–phytase A consisted largely in fat content and in the crumb firmness. The observations provide indirect evidence that in rye bread, amylose-lipid complexes and occlusion of lipids within the gluten network are not as important for a proper bread volume as they are in wheat breads [5]. Structural differences between rye and wheat breads, rather than differences in chemical composition of rye and wheat flours were found to be responsible for reduced in vitro starch digestibility [10]. A continuous phase that forms the crumb of rye bread is created mainly from starch granules, whereas in wheat bread, starch granules are occluded within the gluten network. Furthermore, in rye bread, amylose leaches out and coats the starch granules, what makes the starch resistant to hydrolysis after cooling, forming a less porous, and mechanically firmer crumb structure than in wheat breads [12]. It seems therefore that starch hydrolysis is the main factor contributing to volume of rye bread. In our studies amylolysis was probably stimulated by exogenous phytate-degrading enzymes that freed calcium from complexes with phytate and activated endogenous rye alpha-amylase. The addition of phytase B on top of 3- or 6-phytase A, must have accelerated phytate breakdown additionally what resulted in liberation of starch from complexes with phytate. Autio and co-workers [3] found that the endogenous arabinoxylan-degrading enzyme xylanase in rye flour contributes to the release of amylose from starch granules. From the two preparation of phytase A used in our studies only the 3-phytase A had a significant side xylanase activity. The unique feature of the “Finase P” phytase did not seem to play any role in affecting bread volume. On the other hand, however, the endogenous rye xylanase might have been activated by calcium liberated from its complexes with phytate by the phytate-degrading enzymes. This hypothesis is supported by the fact that calcium protects certain xylanases from proteinase inactivation and thermal unfolding [16].

In spite of slightly reduced moisture, rye breads made with 6-phytase A, and especially with 6-phytase A and phytase B, had significantly improved texture parameters that were retained for at least three days of storage. Texture improving properties of phytases may be related to their ability of releasing calcium from the complexes with phytate that in turn activates endogenous alpha amylase of flour [8, 9] thus generating low molecular weights dextrins that improved crumb firmness. In population of yeast growing during dough fermentation abundant concentration of calcium and myo-inositol phosphates generated by the action of 6-phytase A might result in repressed biosynthesis of membrane phospholipids [1, 6] that had to be synthesised from triacyloglicerols [6, 13], and, consequently, the concentration of fat in breads supplemented with 6-phytase A was decreased by more than 50%. Apparently in breads made with 3-phytase A, the phosphate array on the myo-inositol ring of inositol phosphates was different and the conversion of lipids did not take place. The decreased protein content and slightly elevated content of ash in breads made with microbial 6-phytase A, but not in those supplemented with 3-phytase A, suggest inhibition or repression of yeast growth by metabolites generated by microbial 6-phytase A. In breads baked with phytases A and phytase B, free myo-inositol was possibly produced that could act as an additional repressor of phospholipids biosynthesis [2, 4] in population of yeast growing during rye dough fermentation. Reduced fat content of breads supplemented with pure myo-inositol supports this hypothesis.

Finally, it must be emphasized that the application of 3-phytase A or 6-phytase A in baking technology of wholemeal rye breads results in significantly improved loaf volume. Preparations of phytate-degrading enzymes, and particularly that of 6-phytase A, or 6-phytase A and phytase B, significantly improve crumb texture and exert also a clear antistaling effect. Possible nutritional consequences of using phytates as breadmaking improvers remain to be established.

ACKNOWLEDGEMENTS

Authors would like to acknowledge financial support from the Polish Research Committee (grant Nb. AR 73/31/PBZ/021/P06/99).

REFERENCES

1. Andlid T. A., Veide J., Sandberg A. S., 2004. Metabolism of extracellular inositol hexaphosphate (phytate) by Saccharomyces cerevisiae, Int. J. Food Microbiol., 97, 157-169.

2. Ashburner B. P., Lopez J.M., 1995. Regulation of yeast phospholipid biosynthetic gene expression in response to inositol involves two superimposed mechanisms, Proc. Natl. Acad. Sci., l 92, 9772-9726.

3. Autio K., Härkönen H., Parkkonen T., Poutanen K., Siika-aho M., 1996. Effects of purified endo-beta-xylanase and endo-beta-glucanase on the structural and baking characteristics of rye doughs, Lebensmittel-Wissenschaft Technol., 29, 18-27.

4. Carman G. M., Zeimetz G .M., 1996. Regulation of phospholipid biosynthesis in the yeast Saccharomyces cerevisiae, J.Biol. Chem, 271, 13293-13296.

5. Carr N.O., Daniels N.W., Frazier P.J., 1992. Lipid interactions in breadmaking, Crit. Rev. Food Sci. Nutr., 31, 237-258.

6. Gadd G. M., Foster S. A., 1997. Metabolism of inositol 1,4,5, trisphosphate in Candida albicans: significance as a precursor of inositol polyphosphates and in signal transduction during the dimorphic transition from yeast cells to germ tubes, Microbiol., 143, 437-448.

7. Giovanelli G., Polo R., 1994. Formation of fermentation products and reduction in phytic acid in wheat and rye flour breadmaking, Ital. J. Food Sci., 1, 71-83.

8. Haros M., Rossell C. M., Benedito C., 2001. Fungal phytase as a potential breadmaking additive, Eur. Food Res. Technol., 213, 317-322.

9. Haros M., Rossell C. M., Benedito C., 2001. Use of fungal phytase to improve breadmaking performance of whole wheat bread, J. Agric. Food Chem., 49, 5450-5454.

10. Juntunen K.S., Laaksonen D.E., Autio K., Niskanen L. K., Holst J. J., Savolainen K. E., Liukkonen K-H., Poutanen K.S., Mykkänen H.M., 2003. Structural differences between rye and wheat breads but not total fiber content may explain the lower postprandial insulin response to rye bread, Am. J. Clin. Nutr., 78, 957-964.

11. Leon A.E., Duran E., De Barber B., 2002. Utilization of enzyme mixtures to retard bread crumb firming, J. Agric. Food Chem., 50, 1416-1419.

12. Liukkonen K-H., Juntunen K.S., 2003. Food structure and its relation to starch digestibility and glycaemic response, In: Fischer P, Marti I, Windhab E.J. eds. Proceedings of the 3^rd International Conference of Food Reology and Structure, Zurich, Switzerland.

13. Murphy D. J., 2001. The biogenesis and function of lipid bodies in animals, plants and microorganisms, Prog. Lipid. Res., 40, 325-438.

14. Porres J.M., Etcheverry P., Miller D.D., Lei X.G., 2001. Phytase and citric acid supplementation in whole-wheat bread improves phytate-phosphorus release and iron dialyzability, J. Food Sci., 66, 614-619.

15. Shim J-H., et al., 2004. Improvement of cyclodextrin glucanotransferase as an antistaling enzyme by error-prone PCR, Prot. Engin., 17, 205-211.

16. Spurway T.D., Morland C., Cooper A., Sumner I., Halzewood G.P., O’Donnel A.G., Pickersgill R.W., Gilbert H.J. 1997. Calcium protects a mesophilic xylanase from proteinase inactivation and thermal unfolding, J. Biol. Chem., 272, 17523-17530.

17. Türk M., Sandberg A.S., 1992. Phytate degradation during breadmaking: effect of phytase addition, J. Cereal Sci., 15, 281-294.

18. Ward N. E., 2000. Phytase in commercial poultry diets: an update, In: Proceedings of the 62nd Cornell Nutrition Conference, Ithaca, New York, 8-21.

19. Żyła K., Gogol D., 2002. In vitro efficacies of phosphorolytic enzymes synthesized in mycelial cells of Aspergillus niger AbZ4 grown by a liquid surface fermentation, J. Agric. Food Chem., 50, 899-905.

20. Żyła K., Mika M., Gambuœ H., Nowotny A., Szymczyk B., 2005. Fungal phytases in wholemeal breadmaking.I: 3-Phytase A improves storage stability and in vitro nutrients digestibility of wheat breads, Electronic Journal of Polish Agricultural Universities.

21. Żyła K., Mika M., Stodolak B., Wikiera A., Koreleski J., Œwištkiewicz S., 2004. Towards complete dephosphorylation and total conversion of phytates in poultry feeds, Poultry Sci., 83, 1175-1186.

---

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

---

Responses to this article, comments are invited and should be submitted within three months of the publication of the article. If accepted for publication, they will be published in the chapter headed 'Discussions' and hyperlinked to the article.

---

Main  -  Issues  -  How to Submit  -  From the Publisher  -  Search  -  Subscription