Research Papers on Phytic Acid - 2

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Original article

Traditional sour dough bread (Difo Dabbo) making: I. Effects on phytic acid destruction

Kelbessa Urga¹, Narasimha HV ²

Abstract:  The quantitative changes of phytate during the preparation of the traditional sour dough bread (Difo dabbo) and yeast-raised bread were investigated. Raw materials chosen for investigation were flour of high extraction, soy-fortified wheat flour (Dubbie flour), and white flour. The content of phytic acid was determined in all components (raw materials), intermediate products (doughs), and bread. It was found that pH was the most important factor in reducing phytic acid content. The most marked phytate reduction of 96%-100% occurred in bread made with soy-fortified wheat and white flour sour doughs. Reduction of phytate content in bread made from wholemeal wheat flour sour dough was relatively low. The phytate content in yeast-raised bread was reduced at most to 39% of the initial amount. The study results showed that it should be possible to bake traditional sour dough bread (Difo dabbo) from wholemeals with a low phytic acid content by using the sour dough procedure. Such traditional sour dough bread with very low levels of phytate may be a good source of iron, calcium, and zinc since phytate is known to interfere with the absorption of these minerals.  [Ethiop. J. Health Dev. 1998;12(3):167-173]

Introduction

The preparation of sour dough is one of the oldest biochemical processes used for producing food. Traditionally, sour doughs have been used to produce many types of bread. Although the primary purpose of the sour dough is leavening by yeasts, a simultaneous souring action takes place due to the activities of the lactic acid bacteria present (1). The acidification by sour dough lactic acid bacteria results in bread with a good grain, an elastic crumb and, usually, the characteristic sensory quality of sour dough bread (2). Sour dough fermentation involves a considerable number of heterogeneous metabolic and fermentation reactions which constitute a complicated biological system.

Sour dough bread (locally known as Difo dabbo) occupies a prominent place in the Ethiopian diet and continues to play an important socio-economic role  and  Ethiopian families would not like to pass a single holiday without it. The exact origin of sour dough in Ethiopia is, however, unknown.

The main raw materials employed are wheat supplemented with tef (Eragrostis tef) flour or barley flour. The bread may also be made of dark wheat flour, or from a mixture of various types of flour of consistently higher extraction rate. The method used to prepare a sour dough bread in Ethiopia is largely of empirical origin evolved over a long period.

Traditionally, sour dough bread is produced in Ethiopia by a spontaneous and largely uncontrolled fermentation process. The method of production of Ethiopian sour dough bread resembles the "Sur levain" method of French bread preparation (3).  It differs in the secondary fermentation stage where the sour dough is freshened by additional flour and water and left to ferment for another 3 - 6 hr before baking. The Ethiopian sour dough bread like "Levain" bread is characterised by a marked acidic taste and characteristic aroma. Wheat sour dough is used in France and Italy (4).

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¹From the Ethiopian Health and Nutrition Research Institute, P.O.Box 5654, Addis Ababa, Ethiopia; ²Central Food Technological Research Institute, Mysore 570013, Mysore, India

Cereals (including wheat) are among the main dietary sources of phytate (myo-inositol hexaphosphate) which have inhibitory influence on mineral availability in the gut, notably calcium, magnesium, iron, and zinc (4). The amount of phytate in the diet is, therefore, of practical importance in relation to minerals nutrition on diets based on cereals. The loss of phytate during normal food preparation therefore deserves investigation.

Previous studies on the loss of phytate during bread making have been reported with reference to yeasted and rye sour dough breads (5-7). However, phytate reduction during Ethiopian sour dough bread (Difo dabbo) preparation and the biochemical changes during the fermentation process have not been studied earlier.

This article reports results of a study of the effect of sour dough fermentation on phytic acid degradation in soy-fortified wheat flour, wholemeal wheat flour and white flour breads. We also report on the reduction of phytic acid content in yeast-fermented bread.

Methods

Ingredients:  Soy-fortified wheat flour obtained from Faffa Foods Factory, Addis Ababa, Ethiopia, was transported to India and stored at 4°C until used. Commercial wholemeal wheat flour (72% extraction) and white flour (62% extraction) were kindly supplied by the Department of Milling and Baking Technology, Central Food Tech-nological Research Institute, Mysore, India.

Ersho (starter) preparation:  Ersho was prepared by incubating the flour and double distilled water (40:60, w/w) at 30°C for two days at the start of each fresh ersho preparation. The fermentation culture was kept viable by recycling 10% of the fresh dough with incubation at 30°C for 24 hr. Traditionally, this type of fermentation technology is widely practised in households in Ethiopia. The recycled ersho had a pH of 3.6 to 3.8 and this is referred to as starter culture.

Breadmaking:  Sour dough breads were prepared from soy-fortified wheat flour, whole wheat flour, and white flour. The processing steps in sour dough bread production are illustrated in Figure 1. The sour doughs were fermented at 30°C for 18 hr during the initial phase of fermentation and then six hr in the second phase of fermentation prior to baking with occasional kneading every six hr for five min. The final dough was baked 25 min at 225°C in the Department of Milling and Baking Technology. Straight-dough bread was baked as pup loaves according to the procedure of AACC (10). The dough contained the following ingredients: soy-fortified flour, 300 g; double distilled water, 186 g; sugar, 7.5 g; fat, 6 g; compressed yeast, 6 g; and barley malt flour, 1.5g. The doughs were mixed to optimum in a Hobart mixer, fermented (90% rh) for 170 min at 30°C with 55 min proofing at 30°C, and baked for 25 min at 220°C as described earlier. At the end of each fermentation and baking, the samples were oven-dried at 65°C to a constant weight and ground in an electric grinder (M/S Milone, Rajkot, India) using 0.5mm sieve.

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Analytical methods:  For phytic acid estimation, the samples were extracted with 0.2N HCl for three hr with continuous shaking in a mechanical shaker at room temperature. Phytic acid in the extract was estimated colorimetrically (11). Phytate phosphorus was derived by using the following formula (12):

          A x 28.18

Phytate phosphorus (mg)   =   ------------------

                                            100

where A= phytate content.

Inorganic phosphorus in the sample was extracted in double distilled water by shaking at room temperature for three hr. Inorganic phosphorus in the extract was determined colorimetrically (13). Total phosphorus was determined colorimetrically (13) after digesting the samples in diacid mixture (HNO₃:HClO₄, 5:1, v/v). The pH of doughs and breads was measured with a pH meter in a mixture of ground dry sample (5.0 g) in double distilled water (100 ml). Titratable acidity was determined by titration of 50 ml of filtrate used for pH estimation against 0.1 N NaOH with 1% phenolphthalein indicator. Titratable acidity was expressed as percent lactic acid.

Results were analysed by analysis of variance and means compared by the use of Duncan's multiple range test at the 5% level of probability (14).

Results

Table 1 shows fermentation characteristics of soy-fortified wheat flour sour dough and bread. In the first phase of fermentation, the pH of the dough decreased significantly (p<0.05) compared to the unfermented flour; it dropped to 3.76 after 18 hr fermentation. Addition of flour and water to the fermented

dough increased the pH of the dough due to the buffering action of the flour. The pH, however, decreased significantly (p<0.05) during the second phase of fermentation and baking of bread. With the drop in pH, a concomitant rise in titratable acidity was also observed in both phases of fermentation.   Wholemeal wheat flour fermented for 18 hr had slightly higher pH and titratable acidity (Table 2) compared to the soy-fortified wheat flour (Table 1) and white flour (Table 3) doughs fermented for similar period. White flour and soy-fortified wheat flour doughs, however, had similar pH and titratable acidity values following initial and second phase of fermentation.

In the straight dough process, the pH and titratable values of the dough at 0 hr fermentation were 6.1 and 0.41%, respectively (Table 4). The pH of the dough after declining to 5.76 during 90 min fermentation time remained essentially constant throughout the remaining fermentation period while the titratable acidity of the final dough increased by 1.6-fold. The baking process has slightly increased the pH and decreased titratable acidity of the final loaf.

The total, inorganic and phytate phosphorus content of the sour dough and bread prepared from soy-fortified wheat flour are shown in Table 1. The phytate and inorganic phosphorus amounted to about 53% and 19%, respectively, of the total phosphorus of the  flour.  The concentration of phytic acid (expressed as phytate phosphorus) in wholemeal wheat flour (Table 2) and white

Table 1: pH, titratable acidity (TA), inorganic, phytate and total phosphorus in soy-fortified flour sour dough and bread^*

Time(hr)	pH	TA%	Total P mg/100g	Inorganic P mg/100g	Phytate P mg/100g
Phase I
0	5.91±0.17a	0.41±0.01a	275.13±5.21a	50.91±6.14a	150.05±1.27a
6	4.20±0.02b	0.65±0.04b	275.42±3.34a	95.17±2.31b	102.22±0.98b
12	3.95±0.03c	0.77±0.06c	274.67±1.43a	200.74±5.24c	66.11±0.81c
18	3.76±0.04d	1.12±0.08d	276.09±2.87a	233.62±7.42d	37.78±1.03d
Phase II
0	3.91±0.03b	0.93±0.05e	551.93±7.21b	284.13±3.51e	183.81±2.23f
3	3.83±0.02d	1.13±0.04d	552.87±6.31b	393.36±8.24f	73.43±3.27g
6	3.76±0.06d	1.15±0.04d	551.28±9.17b	430.76±1.84g	36.11±0.72d
Bread	4.17±0.07b	0.95±0.02e	551.89±6.32b	449.82±5.15h	17.78±0.46e

^*Mean values ± SD of three determinations. Values within the same column followed by different siperscript letters are significantly different (p<0.05).

Table 2: pH, titratable acidity (TA), inorganic, phytate and total phosphorus in wholemeal wheat flour sour dough and bread^*

Time        (hr)	pH	TA	Total P	Inorgani P	phytate P mg/100g
Phase I
0	5.13±0.01a	0.52±0.05a	317.80±7.18a	57.16±1.23a	195.38±2.15a
6	4.12±0.04b	0.57±0.02a	319.12±1.32a	86.12±1.23b	12.31±5.04b
12	3.92±0.06c	1.38±0.03b	317.54±4.35a	186.21±4.01c	81.12±2.21c
18	3.83±0.05d	1.80±0.07c	318.87±3.67a	235.51±1.93d	91.02±7.30d
Phase II
0	3.92±0.07c	1.41±0.07b	632.97±5.63b	333.51±1.93e	244.38±2.21e
3	3.83±0.05d	1.75±0.08c	635.13±9.23b	470.57±2.75f	108.73±4.76f
6	3.83±0.06d	1.81±0.05c	643.25±7.57a	535.71±4.45g	43.34±5.27g
Bread	4.02±0.02f	1.79±0.07c	634.34±7.23a	538.11±1.07e	30.42±5.22f
Phase I	5.13±0.01a	0.52±0.05a	317.80±7.18a	57.16±1.23a	195.38±2.15a
0	4.12±0.04b	0.57±0.02a	319.12±1.32a	86.12±1.23b	12.31±5.04b

^*Mean values ± SD of three determinations. Values within the same column followed by different superscript letters are significantly different (p<0.05).

flour (Table 3) were 61.5 and 70%,and 18 and 28%, respectively, of the total phosphorus.

In the fermentation process, the phytate phosphorus content of soy-fortified wheat flour dough was reduced by 81% within 18 hr (Table 1). However, addition of flour to the fermented dough has increased the phytate phosphorus content which later decreased to 91% during the second phase of fermentation (Table 2). The major reduction in phytate phosphorus occurred during the first 12 hr of the initial phase of fermentation in the pH range of 5.91 to 3.95. The rate of phytate degradation was, however, higher in the second phase of fermentation.

In the initial phase of fermentation for wholemeal wheat flour dough, there was a 75% decrease of phytate (Table 2). In the second phase of fermentation this value increased to about 85%. Baking the bread also further reduced the phytic acid by about 3%. Phytic acid in white flour dough was reduced by 88% during the initial phase of fermentation (Table 3). However, no phytate was detected

following second phase of fermentation and baking into bread.

In the straight-dough process, the phytate phosphorus was lost linearly with time during the first 145 min of fermentation period.  After this when loss of phytate had reached 35%, the rate of loss of phytate had declined, so that loss of phytate phosphorus after 225 min of fermentation was only 39%. The rate of loss of the latter was most rapid between 90 and 145 min fermentation period and pH of 5.76 to 5.75 (Table 4).

Losses of phytate phosphorus were followed by increases in inorganic phosphorus. During the first 6 hr of initial phase of fermentation, the soy-fortified wheat flour dough piece gained 45mg of inorganic phosphorus per 100g dough (Table 1). The inorganic phosphorus in the dough increased by about 4.6-fold following 18 hr fermentation. Increase in inorganic phosphorus was 1.6-fold at the end of the second phase of fermentation and baking of bread.

Table 3: pH, titratable acidity (TA), inorganic, phytate and total phosphorus in white flour sour dough and bread^*

Time    (hr)	pH	TA mg/100g	Total P%	Inorganic P mg/100g	Phytate P mg/100g
Phase I
0	5.84±0.11a	0.37±0.07a	83.40±4.56a	23.11±2.17a	58.13±0.28a
6	5.72±0.09b	0.39±0.10a	83.12±2.54a	30.26±1.71b	50.06±0.19b
12	3.91±0.07c	0.95±0.30b	82.97±2.31a	61.31±1.15c	17.13±1.21c
18	4.12±0.03d	0.94±0.09b	83.21±6.13a	63.93±1.32c	25.63±4.53d
Phase II
0	3.98±0.05c	0.94±0.09b	167.31±3.59a	92.14±1.82d	67.33±5.27a
3	3.80±0.03e	1.10±0.04c	168.71±4.17a	139.26±2.24e	24.81±5.43d
6	3.77±0.04e	1.25±0.03d	168.37±3.42a	161.74±1.14f	4.35±1.21e
Bread	4.34±0.04d	1.12±0.07d	169.51±7.15a	163.78±0.43f	----

^*Mean values ± SD of three determinations. Values within the same column followed by different superscript letters are significantly different (p<0.05).

Table 4: pH, titratable acidity (TA), inorganic, phytate and total phosphorus in straight-dough bread^*

Time    (hr)	pH	TA mg/100g	Total P%	Inorganic P mg/100g	Phytate P mg/100g
0	6.06±0.02a	0.41±0.02a	275.13±5.23a	65.71±2.51a	158.51±5.72a
90	5.76±0.03b	0.54±0.01b	275.98±2.31a	69.53±2.30a	120.34±2.27b
145	5.75±0.01b	0.643±0.03c	275.78±5.43a	74.48±4.13b	102.72±3.58c
170	5.78±0.04b	0.67±0.04c	274.76±1.35a	78.86±3.18c	98.75±1.53c
225	5.77±0.03b	0.68±0.02c	2.75.18±8.31a	81.35±2.15c	97.22±0.83c
Bread	5.83±0.03b	0.58±0.05b	274.98±2.24a	84.84±4.39d	96.78±1.68c

^*Mean values ± SD of three determinations. Values within the same column followed by different superscript letters are significantly different (p<0.05).

In wholemeal wheat flour fermentation, the inorganic phosphorus was increased by about 4.6-fold during the initial phase of fermentation  (Table 2).   During  the  second phase of fermentation and baking of the bread, the inorganic phosphorus content was increased by 1.6-fold. In white flour the percentage increase in inorganic phosphorus was about 2.8-fold, after 18 hr fermentation (Table 3). The inorganic phosphorus in the final dough and bread was increased by 1.8-fold. In the straight-dough process, there was only a 24 % increase in inorganic phosphorus following 225 min of fermentation (Table 4). The inorganic phosphorus content of the bread was 31% of the total phosphorus.

Discussion

Ethiopian sour dough bread preparation is a two-phase process in which the mother dough obtained during the initial phase of fermentation is freshened by additional flour and water followed by a second phase of fermentation which usually lasts 6 hr before baking. A number of biochemical changes take place as a result of such fermentation processes. During the initial phase of fermentation of sour doughs, the amount of acid produced increased with concomitant drop in the pH. The low pH and higher titratable acidity in the fermented doughs may be due to the production of organic acids by the fermenting microflora. Rapid drop in pH with a corresponding increase in titratable acidity have been reported during the preparation of sour wheat bread (15).

In the initial phase of fermentation for soy-fortified wheat flour dough, there was about 75% decrease of phytic acid.  The decrease of phytate was higher (83%) only in the case of white flour. This phenomenon might be caused by the use of low phytate raw material for this phase.

In spite of long fermentation time (18 hr) and acidity favourable for phytase activity (pH) the high amount of phytate in wholemeal wheat and soy-fortified flour could not be destroyed as it was the case with white flour. The slow hydrolysis of phytic acid in wholemeal wheat flour fermentation observed in the present study may be due to presence of high concentration of phytate phosphorus in the medium which inhibits phytase activity. This confirms earlier observations made for several sorts of Polish bread and wholemeal wheat flour (9, 16).

As compared with the initial phase, hydrolysis of phytate in doughs in the second phase of fermentation was faster. Addition of the flour to the sour doughs as the pH lowered was the possible reason for increased rate of phytate hydrolysis during the second phase of fermentation.

In the straight-dough process, when breads made with soy-fortified flour, the percentage of phytate hydrolysed in the bread is much lower than in the bread prepared from the sour dough. The major reduction of phytate occurred in the breads during the first 145 min of rising followed by little additional loss up to 225 min of rising. Harland and Harland (1980) observed similar trends in breads prepared from rye flour, white flour, and wholemeal wheat flour (17). In contrast, Ranhotra et al. (1974) observed that more than three-fourth of the phytate was hydrolysed when yeasted breads were made with soy-fortified wheat flour (18).

Ter-Sarkisian et al (1974) compared fermentative loss of phytate in yeasted and sour doughs made from a sample of 75% extraction flour. In two hr at 23°C, flour doughs lost 37-50% of phytate, whereas the yeasted doughs lost only 12-37% (19). Chhabra and Sidhu (1988) in bread made with 1.5% yeast using 85% extraction flour, reported 42-46% destruction of phytate after 3-6 hr of fermentation (20).

The complete hydrolysis of phytate in white flour sour dough observed in the present study confirms earlier observations that all of the phytate in wheat bread was hydrolysed during the process of bread making apparently due to phytase in wheat and/or yeast (17, 21). However, Tangkongchitr et al.(1982) reported that no phytase enzyme is associated with yeast cells as proposed by Harland and Harland (17, 22).

The main controlling factor in phytate hydrolysis appears to be the pH of the dough. A maximum hydrolysis of about 91% and 100%, respectively, of phytate was noted in bread prepared from soy-fortified wheat flour and white flour. The pH of the dough was 3.76. The hydrolysis of wholemeal wheat flour dough bread having a pH value of 3.83 was 88%. This confirms previous observations that  hydrolysis of phytate depends mostly on the acidity and is very low in higher pH values (22). The importance of acidity in doughs was reported by Meuser et al and Bartnik et al (16, 23).

The comparison of the phytate content in the dough after fermentation and in bread indicates that some destruction of phytate takes place during the baking process, when the temperature is still below the inactivating point of phytase. Hydrolysis of phytate in the first stage of the baking process has also been reported previously (16).

The decrease in phytate phosphorus as accompanied by an increase in inorganic phosphorus content in the sour dough fermentation may be attributed to the activity of phytase present in the flour and the fermenting microorganisms as reported in previous studies (6, 17).

The increase in inorganic phosphorus appeared to parallel the breakdown of phytate in sour dough fermentation; an almost quantitative conversion of phytate phosphorus to inorganic phosphorus in dough was noted. All the phytate phosphorus lost during fermentation ended up as inorganic phosphorus in the breads prepared from soy-fortified wheat flour, wholemeal wheat flour, and white flour sour doughs equalled to 73, 90 and 96%, respectively, of total phosphorus.

Thus, it can tentatively be concluded that at no time during fermentation of the sour doughs and baking of bread do any intermediary hydrolysis products accumulate from phytate. Harland and Harland (1980) and Tangkongchitr et al. (1981) observed the same phenomenon which indicates that perhaps all intermediate inositol phosphates in the traditional sour dough breads were dephosphrylated (7, 17).

However, in the straight-dough process, the loss of phytate phosphorus was not equal to the gain in inorganic phosphorus; this indicates that intermediate phosphate esters of inositol accumulated in the dough or bread. Fermentation and baking of yeast-raised bread cause a partial degradation of phytate to simple phosphates and to inositol phosphates with fewer phosphate groups (24). It is possible that these latter phosphates may have similar iron binding properties compared with those of hexaphosphates (25).

This study showed that phytic acid in low extraction flours was more easily hydrolysed than in wholemeal wheat flour. Accordingly, phytic acid levels in breads made from whole wheat meals would be higher than in breads made from white flour. Our results showed that it should be possible to bake traditional sour dough bread with a low phytic acid content by using the sour dough procedure.

This reduction of phytate content to very low levels would make the wholemeal breads a good source of iron, phosphorus, calcium and zinc, since phytate is known to interfere with absorption of these minerals. The presence of ingredients of high phytase activity (e.g. sour dough), particularly in bread containing wholemeal flour is important to achieve effective phytate reduction.

Acknowledgement

This study was financially supported by the United Nations University, Tokyo, Japan and Central Food Technological Research Institute, Mysore, India. The excellent technical assistance of Sasikala BV is gratefully acknowledged.

References

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3.  Seibl W, Brummer JM. The sour dough process for bread in Germany. Cereal Foods World 1991;36:299-302.

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10. American Association of Cereal Chemists. Approved Methods of Analysis of the AACC. The  Association, St.Paul, MN,1969.

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12. Reddy NR, Salunkhe DK, Sathe SK. Phytates in cereals and legumes. Adv Food Res 1982;28:1-92.

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16. Bartnik M, Florysiak J. Phytate hydrolysis during bread making of several Polish bread. Die Nahrung 1988;32:37-42.

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18. Ranhotra GS. Hydrolysis during bread making of phytic acid in wheat protein concentrate. J Food Sci 1972;37:12-13.

19. Ter-Sarkissian N, Azar M, Ghavifekr H, Ferguson T, Hedayat H. High phytic acid in Iranian breads. J Am Diet Assn  1974;65:651-653.

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Original article

Traditional Sour Dough Bread (Difo Dabbo) Making: II. effects on the HCL-extractability of minerals

Kelbessa Urga¹, Narasimha HV ²

Abstract:  Traditional sour dough bread (Difo dabbo) was prepared from wholemeal wheat flour, soy-fortified wheat flour (Dubbie flour) and white flour. Yeast-raised bread was prepared from Dubbie flour by the straight-dough process. Sour dough fermentation of bread significantly reduced phytic acid content and increased the HCl-extarctability of calcium, iron, zinc and phosphorus. The extractability increased with an increase in the period of fermentation. Higher extractability of the minerals was obtained in white flour sour dough bread. Wholemeal wheat flour sour dough bread exhibited relatively lower extractability of the minerals compared to the other two sour dough breads. Significantly (p<0.05) lower values for HCl-extractability of minerals were observed in bread prepared by the straight dough process. The sour dough fermentation is an effective method for improving HCl-extractability and possibly the bioavailability of minerals which helps to prevent and ameliorate mineral deficiencies and improving the nutritional status of people consuming such food. Ethiop. J. Health Dev. 1998;12(3):175-181]

Introduction

Grains and products made from milled grains supply considerable percentages of the nutritionally important proteins, B vitamins, and minerals and trace minerals. Bread, and particularly bread from high extraction flours and whole grain, contains many materials. The higher the degree of extraction of the flour, that is, the greater the yield of flour for a given weight of grain, the greater the amount of seed coat and of the outer layer in the flour. Such flours are darker. Short extraction flour (or white flour) contains almost exclusively portions of the endosperm (1).

The major portion of the minerals is found in the outer layers of the grain, where high extraction flour contains higher concentration of minerals. However, absorption studies in humans indicate that higher extraction flour substantially reduces the availability of  minerals due to the presence of high concentration of phytic acid in the bran (2).

Phytic acid (the hexaphosphate ester of myo- inositol, present in considerable amounts in cereals, has been considered as an antinutrient due to its inhibitory effect on mineral bioavailability. The most striking chemical impact of phytic acid is its chelating ability with multivalent cations, especially divalent and trivalent cations to form cation-phytic acid complexes. The complexes are usually soluble at acidic pH, but they have limited solubility at neutral pH, a pH near to that in the small intestine. The insolubility of the complexes is regarded as a major reason for the reduced bioavailability of phytic acid-mineral complexes (3).

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¹From the Ethiopian Health and Nutrition Research Institute P.O. Box 5654, Addis Ababa, Ethiopia; ²Central Food Technological Research Institute Mysore 570013, Mysore, India

The amount of phytate in the diet is, therefore, of practical importance in relation to mineral nutrition in diets based on cereals. The loss of phytate during normal food preparation, therefore, deserves further investigation. Fermentation is known to bring about several desirable nutritional changes on the fermented products. Previous studies have shown that sour dough and  yeast  fermentation of  bread

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Table 1:  Phytic acid (%) and HCl-extractability (%) of calcium, iron, zinc, and phosphorus  in soy-fortified wheat flour sour dough bread^*

Time    (hr)	Calcium	Iron	Zinc	Phosphorus	Phytic acid
Phase I
0	26.13±1.12a	32.31±3.06a	48.57±0.95a	38.21±0.24a	0.52±0.04a
6	32.42±3.71b	41.26±2.85b	61.72±3.41b	44.41±0.51b	0.35±0.03b
12	40.76±2.34c	49.57±1.30c	65.46±4.41c	51.54±1.19c	0.23±0.03c
18	45.61±4.23d	54.37±2.15d	70.78±5.95d	9.81±0.98d	0.13±0.02d
Phase II
0	39.67±5.76c	49.63±4.58c	73.39±1.41d	64.45±1.87e	0.64±0.08e
3	47.59±1.64d	64.51±3.17e	78.63±2.58e	71.15±1.42d	0.25±0.11c
6	66.41±1.82e	77.12±4.92f	85.71±6.27f	78.18±4.51f	0.12±0.03d
Bread	73.63±7.21f	79.36±5.14f	88.26±2.41f	86.41±4.31g	0.06±0.02e

^*Mean values ± SD of three determinations. Values within the same column followed by different superscript letters are significantly different (p<0.05).

resulted in a significant reduction in phytic acid content (4-6).

Besides reducing the level of phytic acid in the fermented products, fermentation has also been reported to convert bound form of minerals to free forms which is responsible for increased HCl-extractability (an index of their bioavailability to humans) of minerals of the fermented product (7). Solubility of minerals in foodstuffs under simulated gastric conditions has also been reported to be indicative of their bioavailability from those foodstuffs (8).

The effect of natural fermentation on the HCl-extractability of minerals from tef (Eragrostis tef) has been reported previously (9). However, no systematic studies have been carried out so far to evaluate the effect of sour dough and yeast fermentation on the extractability of minerals. This study reports the effect of the traditional Ethiopian sour dough bread (Difo dabbo) and yeast-raised bread making on the HCl-extractability of phosphorus, calcium, iron and zinc in 0.03N HCl, the concentration of HCl in the human stomach.

Methods

Ingredients:  Soy-fortified wheat flour (soy flour, 5%), locally known as Dubbie flour, obtained from Faffa Foods Factory, Addis Ababa, Ethiopia, was transported to India and stored at 4°C until used. Commercial whole wheat flour, 72% extraction, and white flour, 62% extraction, was kindly supplied by the Department of Milling and Baking Technology, Central Food Technological Research Institute, Mysore, India.

Bread making:  Sour dough breads (Difo dabbo) were prepared from soy-fortified wheat flour, whole wheat flour, and white flour as described previously (10).  Yeast-raised bread was baked as pup loaves following the straight-dough procedures as described in the AACC (11). The dough contained the following ingredients: soy-fortified wheat flour, 300 g; double distilled water, 186 g; sugar, 7.5 g; fat, six g; compressed yeast, six g; and barley malt flour, 1.5g. The doughs were mixed to optimum in a Hobart mixer, fermented (90% rh) for 175 min at 30°C with 55 min proofing at 30°C, and baked for 25 min at 220°C as described earlier. At the end of each fermentation and baking, the samples were oven-dried at 65°C to constant weight and ground in an electric grinder (M/S Milone, Rajkot, India) using 0.5mm sieve.

Analytical methods:  Phytic acid contents of the cereal flours and their food products were determined using a spectrophotometer method (12). Inorganic phosphorus in the sample was extracted in double distilled water by shaking at room temperature for three hr. Inorganic phosphorus in the extract was determined colorimetrically (13).

Mineral analysis:  The samples were acid-digested using a nitric acid-perchloric acid mixture [HNO₃: HClO₄, 5:1 (v/v)]. The amounts of iron and zinc in the digested samples were determined by atomic absorption spectrometry (Perkin-Elmer, Model 3110, Norwalk, CT, USA) according to the method of Lindsey and Norwell (14). Phosphorus in the digested samples was estimated colorimetrically (13), whereas calcium was determined by the titration method (15).

Hcl-extractablity of minerals:  The minerals in the fermented and unfermented samples were extracted with 0.03 N HCl by shaking (Environ Shaker, Model 3597-I, LabLine Instruments, Melrose Park, Ill., USA) the contents at 37°C for three hr. The clear extract obtained after filtration with Whatman #42 filter paper was oven-dried at 100°C and wet-digested with diacid mixture. The amounts of extractable phosphorus, iron, calcium, and zinc in the digested samples were determined by the methods described earlier.

Mineral extractable

  in 0.03N  HCl

Mineral extractability (%)=  --------------------------- x 100

    Total mineral

Samples from different fermentation periods were statistically compared using analysis of variance to estimate the level of significance and correlation coefficients according to standard methods (16). Differences were considered significant at <0.05.

Results

In the initial phase of fermentation, the phytic acid contents of soy-fortified wheat flour decreased by 75% (Table 1).  Phytic acid in wholemeal wheat flour (Table 2) and white flour (Table 3) decreased by 75 and 90%, respectively. Addition of fresh flour to the fermented sour doughs increased the phytic acid content which was subsequently decreased to 0.06 and 0.11%, respectively, in soy

fortified wheat flour (Table 1) and wholemeal wheat flour (Table 2) following second phase of fermentation and then baking of bread. Phytic  acid  in  white  flour  was  completely hydrolysed (Table 3) during the second phase of fermentation and baking of bread. In the straight dough process, the overall phytic acid reduction was 39% following fermentation and bread making (Table 4).

Sour dough fermentation significantly (p<0.05) improved the HCl-extractability of phosphorus, calcium, iron, and zinc; the longer the period of fermentation, the higher was the HCl-extractability. HCl-extractability of phosphorus in soy-fortified wheat flour sour dough increased gradually with an increase in the period of fermentation, i.e, from 0-18 hr (Table 1). Similarly HCl-extractability of, phosphorus increased significantly (p<0.05) in wholemeal wheat flour (Table 2) and white flour (Table 3) following initial phase of fermentation. At the start of the second phase of fermentation, the percentage extractability of phosphorus was decreased by about 5%, 8% and 7% in soy-fortified wheat flour (Table 1), wholemeal wheat flour (Table 2), and white flour (Table 3), respectively.  This percentage was increased to about 86, 78 and 87% in soy-fortified wheat flour, whole wheat flour, and white flour sour dough bread, respectively. A significantly (p<0.05) negative correlation occurred between the phytic acid and extractable phosphorus. The extractable phosphorus in the straight-dough bread, however, was low (73%) (Table 4).

Table 2:  Phytic acid (%) and HCl-extractability (%) of calcium, iron, zinc, and phosphorus  in wholemeal wheat flour sour dough bread^*

Time    (hr)	Calcium	Iron	Zinc	Phosphorus	Phytic acid
Phase I
0	21.47±0.82a	28.62±1.47a	44.93±0.14a	34.64±1.19a	0.68±0.07a
6	25.62±0.63a	30.19±2.71a	48.94±0.28b	48.89±0.44b	0.43±0.07b
12	32.71±0.41b	43.24±1.42b	54.15±0.70c	56.11±0.37c	0.28±0.08c
18	46.38±0.87c	46.17±5.78b	61.78±0.68d	62.66±0.88d	0.17±0.06d
Pahse II
0	41.18±0.46d	51.14±1.17c	66.34±1.62e	54.93±0.93c	0.85±0.08e
3	58.67±3.81e	56.84±2.44d	66.51±2.58e	65.27±6.72d	0.38±0.05f
6	61.65±1.62f	66.92±3.27e	70.48±3.94f	75.36±1.45e	0.15±0.05d
Bread	66.72±1.78g	68.11±2.43e	72.76±6.17f	78.37±2.87f	0.11±0.05g

 

^*Mean values ± SD of three determinations. Values within the same column followed, by different superscript letters are significantly different (p<0.05).

Table 3:  Phytic acid (%) and HCl-extractability (%) of calcium, iron, zinc, and phosphorus in white flour sour dough bread^*

Time    (hr)	Calcium	Iron	Zinc	Phosphorus	Phytic acid
Phase I
0	37.62±2.43a	45.78±0.49a	43.65±2.65a	51.33±1.20a	0.20±0.01a
6	45.17±1.71b	51.89±0.89b	49.63±3.88b	54.41±2.48a	0.17±0.01b
12	51.86±0.85c	57.36±0.45c	54.60±3.52c	62.46±1.21b	0.06±0.01c
18	59.78±0.93d	64.21±0.61d	59.54±1.69d	75.28±4.24c	0.02±0.01d
Phase II
0	55.82±2.19c	54.58±0.93b	64.64±2.14d	68.65±5.18d	0.23±0.05a
3	60.65±5.36d	69.62±1.16e	68.56±4.64e	79.31±3.45c	0.09±0.01c
6	72.71±4.46e	77.73±4.34f	72.46±7.03f	85.17±2.69e	0.02±0.01d
Bread	74.15±5.09e	83.69±3.27g	86.16±3.59f	87.49±6.54e	------

^*Mean values ± SD of three determinations. Values within the same column followed by different superscript letters are significantly different (p<0.05).

The calcium extactability increased to varying extent depending upon the period and type of fermentation. Following initial phase of fermentation, calcium extractability increased by 45, 46 and 59% in soy-fortified wheat flour (Table 1), wholemeal wheat flour (Table 2), and white flour (Table 3), respectively. The extractable calcium was lower in wholemeal wheat flour bread (Table 2) compared with the breads prepared from soy-fortified wheat flour (Table 1) and white flour (Table 3). In the straight-dough process, the extarctability of calcium was much lower (only 62%) compared to the sour dough process bread.

The unfermented soy-fortified wheat flour (Table 1), wholemeal wheat flour (Table 2) and white flour (Table 3) water mixtures contained 32, 28 and 46% extractable iron, respectively. Fermentation of the mixture resulted in a significant (p<0.05) increase in the extractability of iron. The extractability increased by about 69% in wholemeal wheat flour (Table 2),72% in soy-fortified wheat flour (Table 1) and 46% in white flour  (Table 3) following the initial phase of fermentation. The extractability of iron reached a maximum after six hr of second phase of fermentation and baking; iron extractability in white flour sour dough breads was significantly higher than in soy-fortified wheat flour and white flour. Bread prepared by the straight dough process has only 56% extractable iron (Table 4).

The HCl-extractability of zinc from the soy-fortified wheat flour (Table 1), wholemeal wheat flour (Table 2), and white flour (Table 3) was 49, 45 and 43%, respectively, at zero hour which increased to 71%, 62% and 60%, respectively, following fermentation for 18 hr (initial phase of fermentation). Addition of flour to the sour doughs and fermentation for sixhr and baking further improved the extractability of zinc. The highest extractability of zinc (88%) was observed in sour dough bread prepared from white flour (Table 3). The extractable zinc in bread prepared by the straight-dough process was low and only 55% (Table 4).

Discussion

Sour dough fermentation of bread resulted in a significant reduction in phytic acid content compared with the yeast-raised bread. The presence of ingredients of high phytase  activity (sour dough) in the fermenting mixtures may have significantly contributed to achieve effective phytate reduction.

With regard to availability, phosphorus in plant materials is much less available than in animal materials as a substantial proportion is originally bound in the form of phytate phosphorus.  In the present study, the improvement in the phosphorus extractability corresponded with a proportional decrease in phytic acid at all periods of fermentation in both the sour dough and straight-dough processes.  This showed that hydrolytic reduction of phytic acid during sour dough fermentation and yeast fermentation may contribute towards increase in the extractable-phosphorus.  Correlation coefficients showed a significant (p<0.05) negative correlation

Table 4:  Phytic acid (%) and HCl-extractability (%) of calcium, iron, zinc and phosphorus in soy-fortified wheat flour yeast-raised bread^*

Time   (min)	Calcium	Iron	Zinc	Phosphorus	Phytic acid
90	35.84±4.38b	36.48±1.28a	36.41±1.32a	44.23±0.29b	0.42±0.07b
145	44.73±2.81c	40.60±3.84b	43.55±2.05b	55.91±0.84c	0.36±0.02c
170	53.47±3.12d	44.19±0.97b	50.12±3.14c	63.85±1.04d	0.34±0.05c
225	58.20±1.95e	52.65±4.15c	52.35±1.68c	67.02±0.76d	0.34±0.03c
Bread	62.72±5.12f	55.54±1.93c	55.25±1.76d	73.66±1.19e	0.33±0.06c

^*Mean values ± SD of three determinations. Values within the same column followed by different superscript letters are significantly different (p<0.05)

between phytic acid and extractable phosphorus.  Thus, the lower the phytic acid, the greater was the extractable phosphorus in the breads.

The reduction in phytic acid during sour dough and yeast fermentations may be attributed to the hydrolysis of phytic acid by phytase.  Phytase, a normal constituent of wheat grain, becomes active in appropriate pH conditions (17).  Phytase may also be produced by the fermenting microflora (18).  Phytase from these two sources hydrolyses the phytic acid in the fermenting mixtures and liberates inorganic phosphorus (values not shown) which may increase the HCl-extractability of phosphorus in the fermented product.  A decrease in phytic acid and simultaneous increase in phosphorus extractability of pearl millet has been reported previously during natural fermentation (19).

The effect of reduction of phytate from the yeast used in yeast-fermented bread can be regarded as minor since, according to previous investigators, it does not contain any or very little phytase (4).  It seems likely that the main phytate reduction in yeast-raised bread was as a result of endogenous phytase activity of the soy-fortified wheat flour.

Sour dough fermentation processes improved the extractability of minerals, an index of their bioavailability to the human system.  The low extractability of minerals during the first six hours of initial phase of fermentation may be ascribed to the high phytic acid content and pH of the fermenting mixtures.  Champagne and Phillipy (1989) observed that the solubility of minerals decreases, and the binding of minerals and phytic acid increases, with increasing pH (20); therefore, it is expected that pH influences the extractability of minerals.

Addition of flour to the fermented dough led to a significant increase in phytic acid content.  This again led to a decrease in the extractability of calcium, iron, and phosphorus at the start of the second phase of fermentation. Hallberg et al.(1987) reported that the removal of phytic acid in bran by endogenous phytase significantly increased iron absorption, and the inhibition could be restored to a marked extent by restitution of the phytate content (21). However, despite the rise in phytic acid content, extractable zinc in the sour doughs remained unaffected at the start of the second phase of fermentation.

Highest extractability of calcium, iron, and zinc was obtained in white flour sour dough bread; that of phosphorus in soy-fortified wheat flour, and white flour sour dough breads.  The HCl-extractability of calcium, iron, zinc, and phosphorus in the sour dough bread prepared from wholemeal wheat bread was relatively lower compared to the other two sour dough breads.  McCance and Widdowson (1942) found that bread from high extraction flour reduced the retention of minerals in humans, and that phytic acid destruction improved their retention (2).  In the prsent study, lower values for HCl-extractability minerals were observed in bread prepared by the straight-dough process.

Higher HCl-extractabilities of calcium, iron, and zinc from the breads prepared from soy-fortified wheat flour, whole wheat flour, and white flour may be partly ascribed to the decreased content of phytic acid, which had a significant negative correlation with the minerals extractability.  Decrease in phytic acid content possibly through hydrolysis by flour phytase and phytase of the fermentative microflora (18) may indicate that the divalent cations are freed from the phytate-mineral complex, which may account for the increased HCl-extractability in the sour dough and yeast-raised breads.  Fermentation has also been reported to increase the HCl-extractability  of minerals in corn and soybean (22-23).

Fermented foods such as bread, contain considerable quantities of inosotol phosphates with less than six phosphates. The dephosphorylation of phytate to lower inosotol phosphates (which might eliminate the negative effect on mineral absorption) may be achieved by fermentation during dough making. In the present study, the decrease in phytic acid appeared to parallel the increase in extractable phosphorous indicating that at no time during fermentation of the sour doughs and baking do any intermediary hydrolysis products accumulate from phytate and perhaps all intermediate inositol phosphates were dephosphorylated.

Lonnerdal et al. (1989) found that the inhibitory effect of phytate was dependent on the degree of phosphorylation of inosotol (24).  At higher degrees of phosphorylation, calcium and zinc absorption was significantly inhibited, whereas no effect was observed at lesser degrees of phosphorylation.

Consumption of low extraction flours in Ethiopian households is limited due to cost and ease of availability.  The traditional diets including sour dough bread (Difo dabbo) are prepared mainly from high extraction flours which are usually rich in phytic acid and minerals.  Effective reduction of phytic acid in foods prepared from such high extraction flours can be obtained through sour dough fermentation.  Sour dough fermentation thus appears to make the wholemeal foods a good source of calcium, iron, zinc, and phosphorus.

The traditional sour dough bread (Difo dabbo) prepared by the sour dough fermentation of wheat is, therefore, an effective method for improving HCl-extractability and possibly the bioavailability of minerals, thus improving the nutritional quality of wholemeal flours.

The availability of minerals from plant foods such as cereals and legumes is limited due to the presence of antinutrients.  Increased extractability of minerals in wholemeal foods, especially sour dough bread, is particularly important from a nutritional view point.  Consumption of these indigenous fermented products may be useful in preventing and ameliorating mineral deficiencies caused by their limited bioavailability and improving the nutritional status of populations consuming such foods.

Acknowledgements

This study was financially supported by the United Nations University, Tokyo, Japan and Central Food Technological Research Institute, Mysore, India. The excellent technical assistance of Sasikala BV is gratefully acknowledged.

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