Phytate degradation during breadmaking: Effect of phytase addition

Journal of Cereal Science 15 (1992) 281-294

Phytate Degradation During Breadmaking: Effect of Phytase Addition M. TURK and A.-S. SANDBERG Chalmers Universzty of Technology, Department of Food Science, cj 0 SIK, Box 5401, S-402 29 G8teborg, Sweden. Received 7 May 1991

Bread was made using whole wheat flour and flour of 60 % extraction, and inositol hexaphosphate and its hydrolysis products were measured by high-performance liquid chromatography (HPLC) in both doughs and breads. Addition of a phytase preparation from A. niger to the doughs resulted in an increased degradation of phytate. When milk was included in the dough formulation, phytate degradation was inhibited almost completely, but fermented milk had no effect. Lactic acid, whether in the presence or absence of calcium chloride, inhibited phytate degradation, although when phytase was added phytate hydrolysis was complete.

Introduction The low absorption of iron and zinc from cereal-based meals has been ascribed to the high content ofphytate (myo-inositol hexaphosphate), which forms insoluble complexes with these minerals at physiological pH values, thereby inhibiting absorption l • Recent studies have indicated that the absorption depressing effect for iron is also pronounced at low levels of phytate 2 • Under optimal conditions for the endogenous phytases of cereals, it is possible to hydrolyse phytate almost completely and thereby to increase markedly the availability of iron and zinc. This has been demonstrated for food processing steps, such as soaking, malting and fermentation 3- 7 • An alternative to activation of the endogenous enzyme is addition of phytase during food processing. Microbial phytase enzyme preparations are now available commercially making their use in food processing technically feasible. Phytase catalysed reductions in the levels of phytate in wheat flour occur during breadmaking through hydrolysis to phosphate and inositol 8 , o. The extent of the reduction is influence by several factors, such as the extent of extraction during milling of the grain 9 , the leavening time lO and temperature, the acidity of the dough l !, yeast or enzymes added to the dough and the presence of calcium salts ll • 12 . The addition of yeast 10 and sourdough 13 has been claimed to enhance phytate degradation. However, it has been observed that an increased yeast concentration did not increase phytate degradation 9 • Commercial enzymes from wheat (phytase, phosphatase) added to whole wheat flour doughs resulted in a significant reduction ofphytate content in the doughs l4 • Addition of calcium salts to the dough has been claimed to reduce phytate hydrolysis during breadmaking ll . 12. 0733-5210/92/030281 + 14 $03.00/0

© 1992 Academic Press Limited

282

M. TORK AND A.-S. SANDBERG

It was demonstrated recently that almost complete (97 %) hydrolysis of phytate of rye and wheat sourdough bread could be obtained when optimal pH conditions for phytase activity were used5,l5. Similar observations were made for bread baked with rye bran scalded at optimal pH and temperature for phytate degradation. Iron absorption from these sourdough breads in humans was found to be increased to the same extent as that from white wheat bread containing no phytate 5. Consequently, the amounts of iron absorbed from bread baked from whole meal flour, with its high content of iron, would be greater than those from white bread provided the fermentation procedures were optimized. Our previous studies in vitro 16 , in animals 17 and in humans 18 • 19 , indicated that hexaand pentaphosphates of inositol, added separately in a white wheat roll formulation, reduced iron and zinc availability, while tetra-and triphosphates of inositol had no such effect. Foods contain mixtures of different inositol phosphates, however, and they also contain different isomeric forms of these compounds that may interact with each other and with other food components. It is possible therefore that, under certain conditions, inositol tri- and tetraphosphates might affect mineral absorption. This study was undertaken to examine the effect of adding a phytase preparation from A. niger to dough on the hydrolysis of inositol hexa- and pentaphosphates to lower inositol phosphates. The effect of added phytase on phytate hydrolysis was compared with that of endogenous flour phytase and that of yeast phytase. A further objective of the study was to establish whether different ingredients/compounds added to the dough influenced the availability of phytate for enzymic hydrolysis by endogenous or added phytases.

Experimental Raw materials Wholemeal wheat flour, wheat flour with an extraction rate of 60 % (Kungsornen AB, Sweden), dry yeast (Jastbolaget, Sweden), margarine (Milda, vegetable fat 80 %, Margarinbolaget, Sweden), milk (Mjolk, Arla, Sweden), fermented milk (Dofilus, Lactobacillus acidophilus, pH 4·4, ArIa, Sweden), lactic acid (90 %, Fluka Chemie AG, Switzerland) and CaCI 2 • 2H 2 0 were used in the preparation of the breads. The milk contained 3 % fat and 120 mg Cal 100 g. The fermented milk contained O' 5 % fat, 160 mg Cal I00 g and 8·4 % lactic acid. Both kinds of milk were pasteurized, homogenized cow's mik. A phytase preparation from A. niger was provided by ALKO Ltd, Rajamiki, Finland, and was added to the doughs in amounts sufficient to obtain a phytase activity of 1500 PU* Ig flour. Enzyme activities of the phytase preparation were as follows: phytase 28000 PU Iml, amyloglucosidase 375 AGU* IML and acid protease 670 HUT* 1m!. Breadmaking procedure The dough formulation was: flour (900 g), dry yeast (10'2 g), salt (15 g), sugar (15 g), margarine (15 g) and water (500 g or 250 g water and 250 g milk). Water (or water and milk) and fat were heated to 45°C and added to the mixture of flour, yeast, salt and sugar. In doughs to which • Units for enzyme activity. PU: One phytase unit (PU) is the amount of enzyme which liberates, under standard conditions (pH 5'0,37 0c), I nmol of inorganic phosphate from sodium phytate in I min. AGU: one amyloglucosidase unit (AGU) is the amount of enzyme, which liberates 1·0 mg of reducing sugars as glucose from starch under standard conditions (pH 4'8, 60°C, 10 min). HUT: one acid protease unit (HUT) is the amount of enzyme that produces from hemoglobin, in I min under standard conditions (pH 4'7, 40°C), a hydrolyzate whose absorbance at 275 nm is the same as that of a solution containing 1·10 f.Lg/ml of tyrosine in 0·006 M hydrochloric acid.

PHYTATE DEGRADATION DURING BREADMAKING

283

TABLE 1. The effects of endogenous phytase and addition of phytase from A. niger(*) on phytate degradation during dough proofing in doughs made from whole wheat flour (A) and control doughs (B) (i.e. made from a mixture (l: I) of whole wheat flour and white flour) A

Proof time (min) 30+0

30+ IS

30+30

30+90

30+ 120

IPS IP6

IP3 IP4 IPS IP6 IP3 IP4 IPS IP6 IP3 IP4 IPS IP6 IP3 IP4 IPS IP6 IP3 IP4 IP5 IP6

A*

----

Raw materials 0'3±0'04 12'6± 1·0

B

B*

Raw materials 0'2±0'03 7·1±0·7

Dough

Dough

Dough

0'6±0'07 1'8±0'OI 0·7 ±0'03 9·4±0·01

0·9±0·04 0'2±0'06 0'2±0'OJ 6'1 ±0'08 0'4±0'03 0'1 ±O'OJ 0'2±0'Ol S'7±0-1 0-4±0-03 0·1 ±O-Ol 0-2±O'01 6-1 ±O'I 0·2±0·01 0·1 ±0'02 0·1 ±O-02 4'2±O'03 0·2±O 0'2±O'OS 0'1 ±0'02 3'9±0'2

1'2±0'06 1'1 ±O 0'4±Q'03 4·9±0·1 1·1 ±0·06 0'7 ±0-03 0'3±0'02 4'7±0 1·0±0·04 0'5 ± 0'04 0'3±0'1 4'7±0'OS

0·2±0·03 0'09±0 0'08±0 2'7±0'02 0 O'!±O'OI 0·06±0·OI 2·1 ±0'02 0'O6±0 O·04±0 O'07±0 2·0±0·02

0'3±0'03 0'2±0 0'2±0 H±O'I 0'2±0'02 O'2±O O·I±O·OI 3-2±0'2

0'1±0'3 O·03±0·02 O·O6±O·01 1-6±0'OS

1'4±0'07 1·4±0·07 0,5 ±O-Ol %±O'S 1·9±0·06 1'4±0'06 0'S±0'02 8,1 ±0'4 0'8±0'1 O-S±O 0·3±0·02 7·1±0·4

0'5 ±O'I 0·5±O·02 0'3±0'02 6·9±O·2

Dough

0'07±0 0·08±0·02 0·03±0 1'3±O-Q]

Values are J.lmol/g freeze-dried sample and are given as means±s.D. for duplicate samples. IP3-IP6: inositol tri-, tetra-, penta- and hexaphosphate. The sum of IP6 and IP5 was significantly different, at each time-point, from the corresponding dough without addition of phytase (P < 0·0 I). phytase, lactic acid and calcium chloride were added, these ingredients were first suspended in the dough water. The ingredients were mixed for 2 min at 70 rpm and then 5 min at 190 rpm in a 30 I Bjorn Varimixer (MKAB, Sweden) with a lSI bowl. The first proof was at 30°C for 30 min. A dough sample was taken after the first proof, and was frozen and freeze-dried. The rest of the dough was then divided into rolls of about 60 g each, and the second proof was performed in a proving chamber (Elektro Dahlen, Sweden) at 37°C and 80 % relative humidity. Samples were taken out after IS, 30, 90 and 120 min, respectively, during the second proof. Two dough rolls were taken out of the fermentation chamber simultaneously at each time-point. One was baked at 225°C for 10 min and allowed to cool for 1 h at room temperature before being frozen and freeze-dried. The other dough roll was immediately frozen and freeze-dried. The freeze-dried dough and bread samples were ground in a coffee mill (KM 75, Krupps Stiftung & Co. KG, Germany) before analysis. The pH of the doughs was measured with a pH meter (71 pH meter, Beckman, U.S.A.) using a suspension of dough (1 g) in distilled water (10 ml). Seven series of doughs were made:

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M. TURK AND A.-S. SANDBERG

A. Dough made from whole-wheat flour only. B. Dough made from equal amounts of whole wheat flour and white flour (60 % extraction rate). This flour mixture was used in all the following experiments, and is referred to as the 'control' dough. C. Dough to which fermented milk was added. The amount of water was reduced to half and an equal amount (250 g) of fermented milk added. D. Dough with the addition of calcium chloride in amounts corresponding to the composition of fermented milk (44,4 mg Ca/IOO g of flour). E. Dough with the addition of calcium chloride (44'4 mg/IOO g of flour) and lactic acid (2'3 g/IOO g of flour) corresponding to the composition of fermented milk. F. Dough with the addition of lactic acid (2'3 g/lOO g of flour) corresponding to the composition of fermented milk. G. Dough with the addition of milk (250 g).

All the series of doughs were made with and without the addition of phytase. The doughs with added phytase are referred to as A "'-0"'.

Determination of inositol phosphates and minerals in doughs and breads Duplicate samples of freeze-dried ground dough or bread (0'5 g) were extracted with 0·5 M HCl (20 m!) for 3 h. The extracts were centrifuged and the supernatant decanted, frozen overnight and centrifuged again. An aliquot (15 ml) of the supernatant was evaporated to dryness and redissolved in 0'025 M HCI (15 ml). The inositol phosphates were separated from the crude extract by ion exchange chromatography according to Sandberg and Ahderinne 20 . The inositol trio, tetra-, penta- and hexaphosphates were determined by ion-pair C 18 reverse-phase HPLC using formic acid/methanol and tetrabutylammoniumhydroxide in the mobile phase 1o . 2o . The HPLC system comprised an HPLC pump (Waters Model 510, Waters Associates inc., Masschusetls), a C 18 Chromasil (5 Jl) column 2 mm i.d. and a refractive index detector (ERC- 7510 RI-detector Erma Optical Works Ltd., Japan). The flow rate was 0'4 ml/min. Retention times and peak areas were measured by the laboratory data system, HP 1000 (Hewlett Packard Co.). Injections were made with a 20 Jllioop. Samples for analyses of iron, zinc, magnesium and manganese were prepared by dry-ashing of freeze-dried material (0'6 g), and for calcium and phosphorus by wet-ashing. Iron, zinc, magnesium, manganese and calcium were determined against their blanks in an atomic absorption spectrophotometer (Perkin Elmer 360). Phosphorus was determined according to Fiske and Subbarow 21 . Determinations of nitrogen, sodium and potassium were performed as described previously22.

Statistical methods Differences in the mean values of phytate content in doughs and breads were tested statistically by Student's t-test.

Results Phytate contents in raw materials The amounts of inositol trio, tetra-, penta- and hexaphosphates (IPa-IP a) in dry, raw materials were: dough formulation A (whole wheat flour): IPs: Q'3±O'04 and IP o: 12·6 ± 1.0 llffioljg. Dough formulation B, referred to as the' control' dough throughout

PHYTATE DEGRADATION DURING BREADMAKING

285

TABLE II. The compositions of bread rolls made from whole wheat flour (A) and control flour (B) Component

A

B

~-~---

Protein (N x 5'7) g Sodium (mmol) Potassium (mmo!) Calcium (mmol) Magnesium (mmol) Phosphorus (mmo!) Zinc (!-Lmol) Iron (!-Lmol) Manganese (j.tmol)

12·1 to'4 29·9tO·2 12·5±O·3 1'1 to'05 5'1 ±O·O6 11-6tO'04 46'9tO'8 62'3±O-4 26'9tO'8

ll'7tO'2 28·7tO·04 6·6±O·O2 O·9±O·1 2'8±O'OI 8'7tO'4 29·9±O·O2 95'2t 1·5 16'5±O'2

Amounts per lOO g of freeze-dried wheat roll. Values given are the means ± S.D. for duplicate samples. The analyses of minerals were performed at the Department of Clinical Nutrition, Goteborg, Sweden.

this paper (mixture of whole wheat flour and white flour): IP 5 : 0·2±0·03 and IPij: 7-1 ±0'7 Jlmol/g (mean±s.D.). No lower inositol phosphates (IP 3 or IP'I) were detected in the raw materials.

Effects of endogenous phytase on phytic acid during breadmaking The effects of endogenous phytase and proof time on the phytate content of doughs and breads are shown in Table I. A significant proof time-dependent decrease in phytate content was observed in both doughs A and B. After 150 min of fermentation (30 + 120) the inositol hexa- and pentaphosphates were reduced by 44 % (A) and 55 % (B) of the initial value for the raw materials. Initially, inositol, tetra- and triphosphates were not present in the doughs. When inositol hexa- and pentaphosphates were hydrolysed during proofing, the amounts of inositol tetra- and triphosphates rose and then decreased again during the second proof.

Mineral and protein contents of wheat rolls The protein and mineral contents in the wheat rolls are given in Table II. Owing to the fortification of white flour in Sweden with iron (6'5 mg/IOO g), the roll containing white flour had a higher iron content.

The effect of adding A. niger phytase to the dough The effects of adding phytase from A. niger to the doughs are demonstrated in Table I. In the whole wheat dough (A *), phytase addition resulted in a significant proof timedependent decrease in phytate. Up to 69 % of the initial inositol hexa- and pentaphosphates of the raw material was hydrolysed (up to 78 % in the baked bread). A mixture of whole wheat flour and white flour (B) was used in the rest of the experiments, and is referred to throughout as the .control' dough. In this dough, a

286

M. TDRK AND A.-S. SANDBERG

TABLE III. The effects of milk (0), fermented milk (C) and phytase from A. niger(*) on phytate degradation in control doughs

0*

0

Proof time (min) 30+0

30+ 15

30+30

30+90

30+ 120

IP5 IP6

_._--IP3 IP4 IP5 IP6 IP3 IP4 IP5 IP6 IP3 IP4 IP5 IP6 IP3 IP4 IP5 IP6 IP3 IP4 IP5 IP6

Raw materials 0·ltO·03 7-1 to'7 Dough 0'7±0'01 0'6±0'Ol 0'5tO 8·1 ta'09 0'7tO 0·6tO·O] 0·5tO·Ol 7'3tO-01 0'6tO'1 0·StO-03 0'4tO'02 7'2ta'2 a,Sta·os 0-4tO·02 0'4tO-02 6'4tO'06 0'4tO'Ol 0·3 ta'03 0'3tO'02 6·ltO·4

Dough --------_. 0·3 ±0'03 0·6tO·08 0·2±0-01 3-7tO'09 O'2tO-0] O'04tO'0] 0·1 to'O] 3-5tO·06 O·ltO·1 0'4tO 0·1 to'Ol 2'8tO'03 a-2tO'03 0·04tO·0] O-ltO l-7tO'Ol O'ltO'OI 0'5tO'Ol D,] to'05 2'5tO'04

C

C*

Raw materials 0'ltO'03 7-] to'7 Dough 0'S±0'09 0·7tO·08 a·stO'04 4·9±0·2 0·8tO·a4 0·6tO·06 0·5tO·01 4'8tO'4 0·8tO·1 0'5 to'03 0'4±0 HtO'3 0'7±0'05 0'4tO'06 0·3tO·Ol 4·0tO·3 0'6tO'05 0'3tO'Ol 0'3 to'03 3-5tO'3

Dough 0'3 to-1 0'6tO 0'1 to'03 2·7tO·j 0'] to·a1 0'04tO 0-08tO l'OtO'] O·ltO·Ol 0'05tO a·] to·a2 2·8tO·01 0·1 to'02 0'03tO 0'07tO 1·8tO·] 0·1 to·OS 0'03tO 0'07tO'Ol 2'OtO'3

Values are I-lmol/g freeze-dried sample, and are given as means±s.D, for duplicate samples. IP3-IP6: inositol trio, tctra-, penta- and hexaphosphate. The sum of IP6 and IPS was significantly different, at each time point, from the corresponding dough without phytase addition (P < 0-01). Doughs containing fermented milk were significantly different from doughs with milk.

significant decrease in inositol hexa- and pentaphosphate (81 %) was obtained with phytase addition (B*) after 30 + 120 min proof time (up to 88 % in the baked bread). In both dough formulations (A and B), the contents of inositol tetra- and triphosphatcs also decreased when phytase from A. niger was added. Addition offermented milk or milk to the dough _

Milk and fermented milk are sometimes included in dough formulae. As these ingredients contain calcium, phytate hydrolysis might be affected. Table III shows the effects of the addition of fermented milk or milk to the dough. When half the water in the mixture was substituted by milk, phytate hydrolysis was almost completely inhibited. A significant decrease in phytate was observed when phytase was added to the dough

PHYTATE DEGRADATION DURING BREADMAKING

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FIGURE 1. Phytate degradation during breadmaking: effects of milk, fermented control; --,6.--, milk and phytase. -6,-, milk; -G-, fermented milk; milk + phytase; -- 0 --, fennented milk + phytase; - - . --, control +phytase.

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made with milk but the decrease was not as great as that observed when phytase was added to the control dough, Le. without milk. When fermented milk was used, the decrease in phytate was significantly greater than with the same amount of milk, and the values did not differ significantly from those for the control dough without milk or phytase. When phytase was added with fermented milk the dough contained significantly less phytate than dough with milk plus phytase, but the values did not differ significantly from the control dough with added phytase. When phytase was added, the amount of inositol tetra- and triphosphates decreased in doughs with addition of milk and fermented milk. The contents of inositol penta- and hexaphosphates in the baked breads were generally somewhat lower than in the doughs, and are shown in Fig. 1.

288

M. TURK AND A.-S. SANDBERG

TABLE IV. The effects of calcium chloride (D), lactic acid (F) and phytase (*) on phytate degradation during proofing in control doughs

D

Proof time (min) 30+0

30+ IS

30+30

30+90

30+ 120

IPS IP6

IP3 IP4 IPS IP6 IP3 IP4 IPS IP6 IP3 IP4 IPS IP6 IP3 IP4 IPS IP6 IP3 IP4 IPS IP6

D*

Raw materials 0·2±0·03 7·1 ±0·7 -------Dough Dough O·6±O·OI O'5±0'OI O'3±0'02 5'3±0'3 0·6±0·01 0-4±0 0'3±0 4·8±0·02 O'8±0-O6 0'3±0 0'2±0 4·7±0·08 0-4±O'02 O'2±O-01 0·3 ± 0·03 4·2±0·2 0'3±0-04 O'2± 0·01 0·2±0·02 4'0± 0·1

O'3±O 0-2±O 0-2±0'01 4·8±O·03 0·4±0·02 0·1 ±0·02 0·1 ±O 3-8±0·01 0'3±0'OI 0'1 ±O O'I±O 3·4±0·2 0·3±0·01 0·1 ±O 0'1 ±O 3'3 ± 0·1 0·3 ± 0·01 0'2±0 0·1 ±0·01 3·S±0·1

F

F*

Raw materials 0'2±0'03 n±0'7 Dough

Dough

tr. 0-4±O-OI )-1 ±0-02 5·8±0·1 0·2±0·02 0'5 ±0'02 1·3 ±0'03 5-7±0'2 0·1 ±O 0'6±0'04 1·3 ±0'09 5·3±0·4 0-2±0'O3 0·8±O·O4 1'5±O'O8 5·4±0·3 0·2±0·01 0'8±O'02 I'S±0-04 5·2±0·1

j-7±O'02 0'5±O'01 0·3±0 0·8±0·05 0·6±O·1 0'3±0 O'I±O 0'4±0'02 0·8±0·01 0·3±0 0'1 ±O 0'3±O'01 tr. O'O7±0 0 0 tr. 0'04±0'O2 0 0

Values are llmol(g freeze-dried sample, and are given as means±s.D. for duplicate samples. IP3-IP6: inositol tri-, tetra-, penta- and hexaphosphate. The sum of IP6 and IP5 was significantly different from the corresponding doughs without addition of phytase (P < 0'01). All doughs containing lactic acid were significantly different, at each time-point, from the corresponding doughs without lactic acid (P < 0'01). The doughs to which calcium chloride was added were not significantly different from the corresponding doughs without calcium or the doughs with fermented milk.

Effects of adding a soluble calcium salt and lactic acid to the dough Phytate hydrolysis was prevented when milk was added to the dough, but to a lesser extent when fermented milk was used. To determine whether this was an effect of calcium (in milk and fermented milk) or lactic acid (in fermented milk), calcium chloride and lactic acid were added individually and together to doughs. Phytate hydrolysis was not significantly affected compared with the control dough when calcium chloride was added (Table IV). Addition of phytase to the dough containing calcium chloride increased the hydrolysis of phytate significantly. When lactic acid (Table IV) or lactic acid and calcium chloride (Table V) were added to the dough, the reductions in phytate levels were significantly smaller than that in the control dough. In contrast, when both lactic acid and phytase were added, phytate hydrolysis was complete (Table VI). A total

PHYTATE DEGRADATION DURING BREADMAKING

289

TABLE V. The effects of addition of both calcium chloride and lactic acid (E), and phytase (*) on phytate degradation during proofing in control dough

E*

E

Proof time (min) 30+0

30+ 15

30+30

30+90

30+ 120

IP5 IP6

Raw materials 0·2tO·03 7'1 to·7

Dough IP3 IP4 IP5 IP6 IP3 IP4 IPS IP6 IP3 IP4 IPS IP6 IP3 IP4 IP5 IP6 IP3 IP4 IPS IP6

0-2tO-02 0-3tO a'8tO 6·0±0·OS 0'2±0'04 0'4tO I'0±0'04 S·8±0·OS 0-2tO'02 OA±O'OI 1'0±O'02 5·8±0·03 0·2tO·OI 0·5±0·02 1-I±O'06 5'6±O'2 O·2±0·OS 0'5±0 1·1 ±O S'3±O'1

Dough 0'9tO-3 O·3tO·0I O'2±0'01 1·1 ±O'OJ O·6±0·02 0·2±0 0·1 to O'5tO'02 O'4±0'03 O'2tO'01 O·I±O·OI 0·4tO·02 0·1 to·OI 0'06tO 0'02tO 0'06±0 0·1 to 0'05tO-01 0-02tO 0'03 to

Values are J.Imol/g freeze-dried sample and are given !!S means ± S.D. for duplicate smnples. IP3-IP6: inositol tri-, tetm-, pent!!- and hexaphosphate. The sum of IP 6 and IP 6 was significantly different from the corresponding dough without phytase addition (P < 0'01). All doughs containing lactic acid were significantly different at each time-point from doughs without lactic acid. All doughs with calcium chloride and lactic acid were significantly different from the corresponding doughs with fermented milk.

of 99·6 % of the inositol hexa- and pentaphosphates was degraded in the bread containing lactic acid, calcium chloride and phytase after 30 + 120 min of fermentation. In this bread, the inositol tetra- and triphosphates were almost completely degraded. The contents of inositol penta- and hexaphosphates in the baked breads were generally somewhat lower than in the doughs, and are demonstrated in Fig. 2. pHs of the doughs

Since phytase activity is highly dependent on pH 23, the pHs of the doughs were measured and are presented in Table VI.

290

M. TURK AND A.-S. SANDBERG

TABLE VI. Summary of the effects of phytate reduction during breadmaking and change of pH in the doughs during proofing

Flour mixture, additives WWb WW+phytase Control b Control + phytase Control + milk Control + milk + phytase Control + fermented milk Control + fermented mllk + phytase Control + lactic acid Control + lactic acid + phytase Control + calcium chloride Control + calcium chloride + phytase Control + lactic acid+calcium chloride Can trol + lactic acid + calcium chloride + phytase

Change of pH in the doughs"

Phytate reduction in breads" (%) 30+ 15 d

30+30d

30+90 d

30+ l20 d

6'0-5'8 6'0-5·8 5,8-5'7 5,7-5'5 5'9-5·7 5'5-5'3 5'5-5,3 5'5-5,3

35 67 47 82 5 59 39 71

30 69 49 81 10 60 39 75

40 78 60 88

31 78

51 78

88 29 74 51 81

3'4-3-4 3'6-3'6

18 89

16 89

16 98

14 100

5,3-5,4

36

30

51

41

5'4-5'2

44

49

60

60

3·4-3-4

II

14

18

15

3·4-3·5

86

92

98

99-6

29 64

64

" The sum of inositol hexa- and pentaphosphate, calculated on the basis of dry weight. Percentage degradation as compared with initial contents in raw materials. b WW - whole wheat flour was used in the dough. Control - a mixture (1 : 1) of whole wheat flour and white flour was used. C pH values are given for 30 + 15 min and 30 + 120 min of proofing, respectively. d Minutes of proofing.

The pH values were between 5·3 and 6·0 in all doughs except E, E*, F and F* (where lactic acid was added). These doughs had pH values between 3·4 and 3·6. Discussion

All doughs investigated showed a fermentation time-dependent reduction in phytate levels. Most of the degradation occurred during the first fermentation (30 min). During the second fermentation the rate of phytate hydrolysis was considerably lower. This was in agreement with the work of Nayini and Markakis 2 4, who found the greatest decrease in phytate during the first 30 min of dough fermentation. In several other studies9.10.25 it has been shown that the loss of phytate is relatively rapid at the beginning of dough fermentation and that it is not particularly favoured by a prolonged fermentation time. Some destruction of phytate also took place during the baking process whilst the temperature was still below the inactivation point of phytase. The extent of phytate

PHYTATE DEGRADATION DURING BREADMAKING

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FIGURE 2. Phytate degradation during breadmaking: effects of CaCI 2 , lactic acid and phytase. -0-, lactic acid;-.A.-, CaCI 2 + lactic acid ;-0-, CaCI 2 ;--D--, CaCl 2 + phytase; control; -control + phytase; --.. --, CaCI 2 +lactic acid + phytase; -- 0 --, lactic acid + phytase.

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hydrolysis has been shown to be greater in doughs made with flours of lower extraction rate than with whole meal f1our9.25, and this finding was confirmed in the present study. This could be due to product inhibition, which has been suggested by other authors 23 • 26 • In all doughs, the addition of phytase from A. niger resulted in a significantly increased phytate hydrolysis (Table VI). Similar effects on phytate degradation were obtained with another batch of phytase from the same source, which additionally had higher activities of ~-glucanase (400 BUt!m\), amyloglucosidase (4970 AGU Iml) and acid protease (960 HUT Iml). Previous studies have shown that Swedish yeast contains little if any phytase activity27. The yeast used in the present study also contained no phytase activity, and no phytate degradation took place in bread baked from flour in which phytase had been inactivated by autoclaving for 6 min at 120°C (unpublished results). This indicates that the degradation of phytate that occurred in the absence of added enzyme was due to endogenous phytase activity in the flour. t BU: One j}-glucanase unit (BU) is the amount of enzyme which under standard conditions (pH 4·8. SO°C) produces I nmol of reducing sugars as glucose from j}-glucan in one second (1 BU = 1 nkat).

292

M. TURK AND A.-S. SANDBERG

The extent of phytate hydrolysis in doughs that contained lactic acid but no added phytase was less than in the control dough, probably because the endogenous phytase of wheat flour was inactivated owing to the low pH value (3'4). The optimum pH for wheat phytase has been shown to be 5.15 28 • Plant phytases have pH optima at 4,8-5,6, and their activities diminish markedly as the pH is varied from its optimal value 23 • With the addition of lactic acid and A. niger phytase, the dephosphorylation of phytate was rapid and complete even if calcium was present. It is likely that this effect is due to the fact that the pH of the doughs (3'4-3-6) was close to the lower pH optimum of the exogenous phytase, and that calcium did not reduce the solubility ofphytate at this pH. Phytase produced by A. niger has two pH optima: one at 2'5-3,0 (possibly due to additional acid phosphatase activity) and one at 5.0 29 • Microbial phytases also seem to be less sensitive to pH and to be active over a wider pH range than plant phytases 30 - 32 • The addition of hydrochloric acid to wheat flour and adjustment of pH to 3·6 also resulted in complete reduction of phytate when phytase from A. niger was added (unpublished results). Phytate hydrolysis was prevented when milk was added to the dough, but CaCl 2 and fermented milk had no significant effect. There are a few earlier reports indicating that milk and calcium can interfere with phytate degradation during breadmaking. The addition of milk powder 33 • 3,j and dried skimmed milk 35 to doughs was found to inhibit phytate hydrolysis. Ranhotra 12 observed an inhibitory effect of different soluble calcium salts (125 mg Ca/IOO g flour) on phytate hydrolysis during breadmaking. Calcium acetate (146'3 mg/lOO g flour) was found to inhibit phytate degradation completely and calcium phosphate (102-5mg/100 g flour) inhibited it by 50%11. This inhibition was ascribed to the formation of insoluble calcium phosphate complexes. That no significant effect of calcium chloride on phytate degradation was observed here may be due to the lower amount added (44'4 mg Ca/100 g flour). In the present study the addition of milk (33'3 mg Cal 100 g flour) in breadmaking thus depressed phytate hydrolysis to a greater extent than could be accounted for by its calcium content. A similar observation was made by Zemel and Shelef 36 • In the present study phytate hydrolysis due to exogenous A. niger phytase was not inhibited by milk. The degradation of phytate was significantly increased when fermented milk (44-4 mg CallOO g flour), rather than milk itself, was added to the dough and was comparable to that obtained for the control dough. This may be explained by the presence of lactic acid in fermented milk increasing the solubility of calcium phytate by lowering the pH. According to M011gaard 37 , several oxy-acids, including lactic acid, increase the solubility of calcium phytate. Another explanation may be that the pH of the dough with fermented milk (5'5-5'3) was closer to the optimum for the endogenous wheat phytase compared with the dough with milk (5,9-5'7). The use of milk rather than water in breadmaking reduces phytate hydrolysis and may therefore have nutritional implications, such as a negative effect on iron absorption 38 • In this respect, fermented milk seems preferable as an alternative to milk for use in breadmaking. To improve the bioavailability of iron in cereals substantially, phytate degradation must be extensive. As little as 0'5 ~mol/g dry sample of inositol hexa- and pentaphosphates has been shown to reduce iron bioavailability, estimated either in vitro3,16 or in ViV0 39 , when promoting factors are not present. Addition of phytase to the

PHYTATE DEGRADATION DURING BREADMAKING

293

whole meal wheat/white flour bread reduced the phytate content by 88 % to 0·8 I-lmolfg. Although a much lower level than that present initially, this would still be considered sufficient to interfere with iron absorption. When lactic acid was added together with phytase in the present work, more than 99 % of the phytate in the control dough (made from a mixture of whole wheat flour and white flour) was hydrolysed. Microbial (A. niger) phytase may be useful in the production of lactic acid fermented cereals and legumes (which normally have a low pH value) aimed at a high mineral availability. Hydrolysis of phytate during proofing leads to formation of inositol phosphates with different numbers of phosphate groups. In doughs and breads A and B appreciable amounts of inositol tetra- and triphosphates were formed and were partly hydrolysed during the second proof. Generally, however, the amounts ofIP a and IP4 were lower in the doughs and breads with addition of phytase. We found previously that, when added separately to a white bread, inositol tetra phosphate had no effect on zinc and iron absorption 18 • 19 , while inositol penta- and hexaphosphates depressed zinc and iron absorption. However, in processed foods inositol tri- and tetraphosphates may contribute to the impaired absorption in proportion to the number of phosphate groups present. A strong negative correlation between iron and zinc absorption and the sum of inositol tri- to hexaphosphates (mg P) in bread meals has been found5.4°. Further studies are needed, therefore, to understand fully the importance of different inositol phosphates for mineral utilization. Conclusions

If the pH is lowered, it is possible to reduce the phytate content of whole meal wheat bread, by addition of A. niger phytase, to a level not considered to affect iron absorption. When mineral availability is considered, the use of fermented milk may be preferred over milk for use in breadmaking as it had no inhibitory effect on phytate hydrolysis. This study was supported by ALKO Ltd, Finland, and the Swedish Council for Forestry and Agricultural Research (Project no. 599/89 L 135: 2).

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