INTRODUCTION:

Phytic acid (PA), also called myo-initisol hexaphosphate (IP6), is the major phosphorus storage compound in mature cereal and legume grains^1,2. It constitutes l - 2% of the dry weight of many legumes and cereal samples³. About 70% of total phosphorus in plant is bound as phytate phosphorus⁴.

The molecular formula of phytic acid is C₆H₁₈O₂₄P₆. It is highly charged with six phosphate groups. In addition, it includes 12 dissociable hydrogens. The commonly accepted phytic acid structure was defined by Anderson in 1914 ^5,6,7 [Figure (1)]⁶. Moreover, the 12 hydrogens in the phytic acid molecule have different disassociation constant, pKa⁵.

Figure (1): Structure of phytic acid was suggested by Anderson

Phytic acid is well-known as an antinutritional substance, because it's strong ability to chelate multivalent metal ions (specially zinc, calcium and iron), and thus decrease solubility, functionality, absorption and effectiveness^5,8. Most PA–mineral complexes are insoluble at physiological pH, which lowers the bioavailability of minerals⁸.

Mineral bioavailability in grains can be improved by the action of phytase, which hydrolyzes (dephosphorylates) PA to free inorganic phosphate and lower inositol phosphate esters⁹ such as inositol pentaphosphate (IP5), inositol tetraphosphate (IP4), inositol triphosphate (IP3) and possibly the inositol di- and monophosphate during germination, fermentation and food processing⁴. Human or a gastric animals lack sufficient endogenous intestinal phytase which works in the gastrointestinal tract^1,5. In addition, PA is sometimes hydrolyzed by non-enzymatic cleavage¹⁰.

Different processing methods have been known to reduce the level of phytic acid such as roasting, germination, fermentation and baking^10,11,12.

Thus, the goal of this work was to assess phytic acid of peanut and wheat, then study the effect of different domestic processing methods on the phytic acid content of them.

MATERIALS AND METHODS:

Reagents and solutions:

Phytic acid solution (50%) was purchased from Sigma-Aldrich, hydrochloric acid 0.6 N, Wade reagent (0.03% solution of FeCl₃.6H₂O + 0.3% sulfosalicylic acid), 0.1 N sodium hydroxide, 0.1 N potassium acid phthalate.

Samples:

10 samples of peanut, 3 samples of whole wheat and 10 samples of white wheat flour were obtained from many local markets in Damascus.

Equipment:

Spectrophotometer UV-VIS model HTACHI U-1800, centrifuge model Heraeus Christ Labofuge I, pH meter model 420A of Orion, sensitive balance was produced by Sartorius, tubes, pipettes titration, balloons titration.

EXPERIMENTAL PROCEDURES:

Roasting:

The 3 samples of peanuts were divided into three portions that were roasted for (10, 20, 30) min at 150 ^◦C in an electric oven. Whereas the effect of time roasting on PA content of peanuts was studied.

Germination:

The whole wheat seeds were germinated in dishes lined with wet cotton and allowed to germinate for 3 days. Germinated seeds were frozen for 12 h to stop the germination process. After that, the seeds were dried in an electric oven.

Fermentation and baking:

The 3 samples of white wheat flour were divided into four portions that were mixed with distilled water until smooth texture dough. Then the first and second sections were not fermented, whereas the first was not baked but it was dried directly, while the second was baked for 20 min at 180 ^◦C. The third and fourth sections were fermented for 30 min at 38 ^◦C, whereas the third was not baked then it was dried directly while the fourth was baked for 20 min at 180 ^◦C [Table (1)].

Table (1): The sections of samples

Sections	Fermentation	Baking
1	-	-
2	-	+
3	+	-
4	+	+

Germination and processing treatments were replicated three times. Raw and processed samples were ground manually, and then passed through a 0.5 mm sieve.

Phytic acid extraction and determination:

One gram of ground sample was extracted with 20 ml of 0.6 N HCl for 1 h at room temperature with stirring. The extract was centrifuged at room temperature for 20 min and the supernatant was decanted^13,14. A 5 ml aliquot of the supernatant was diluted to 50 ml with distilled water.

The calorimetric assay of phytate was performed according to Latta and Eskin. 1ml of the Wade reagent was added to 3 ml of the assayed solution and the mixture was centrifuged for 5 min. The absorbance at 500 nm was measured using a spectrophotometer^13,14,15.

RESULTS AND DISCUSSION:

Standardization of phytic acid solution:

Phytic acid solutions were used as control samples in establishing the colorimetric method. To standardize phytic acid solutions, a typical acid-base titration was performed with a 0.1 N NaOH solution (0.1 N NaOH was standardization with 0.1 N potassium acid phthalate). To accurately record the pH change, a pH probe was used to obtain a titration curve [Figure (2)].

Figure (2): Acid-base titration curve of phytic acid titrated with sodium hydroxide

The acid-base titration curve showed phytic acid’s deprotonation consists two steps. The first end point (pH ~5.7) was sharp, corresponding to deprotonation of the first six hydrogens with low pKa values. The second end point (pH ~9.5) was not obvious because of weak disassociation of the rest hydrogens. The first end point was apparently a better choice for quantitation. The concentration of this phytic acid solution was determined to be 715.8 g/l. The density of the PA commercial solution was 1.432 g/mL measured using an analytical balance. Therefore, the phytic acid commercial solution was standardized as 50 % w/w.

Validation of the colorimetric method¹⁶:

The pink color of the Wade reagent is due to the reaction between ferric ion and sulfosalicylic acid with an absorbance maximum at 500 nm. In the presence of phytate the iron becomes bound to the phosphate ester and unavailable to react with sulfosalicylic acid, resulting in a decrease in pink color intensity. Whereas the distilled water was used to zero spectrophotometer.

A seven-level standard series was established 0, 5, 10, 30, 50, 70, 90 mg/l for phytic acid. Whereas they were acidification of HCl 0.6 N by 10%. The absorbance at 500 nm was measured. The absorbance is plotted against the phytic acid concentration [Figure (3)].

Figure (3): Line chart of phytic acid with absorbance

Of the graph, the correlation coefficient R² was 0.9991 and the straight-line equation was y = -0.0043x + 0.4529. Thus, the method has a good linearity.

Accuracy should be reported as percent recovery by the assay of known added amount of analyte¹⁶. The percent recovery was 101%.

The standard deviation, relative standard deviation (coefficient of variation) should be reported for precision investigated¹⁶. The standard deviation was 1.18. The relative standard deviation was 2.43%.

1.3 mg/l was determined as the detection limit of phytic acid and 3.9 mg/l was determined as the quantitation limit in this study.

Distribution of phytate in cereal grains:

The phytate contents of cereal grains are shown in Table (2).

The phytate contents of the screened whole wheat ranged from 15.07 to 15.75 mg/g. These values agree with published data between 9.6 and 17.5 mg/g by Garcia-Estepa et al⁴. The phytate contents of the screened peanut ranged from 13.5 to 20.6 mg/g and white wheat flour ranged from 2.71 to 5.44 mg/g. These values agree with published data for peanut between 10.5 and 17.6 mg/g and for white wheat flour between 2.5 and 13.7 mg/g by Reddy¹⁷. The differences in phytate contents of cereal grains may be due to differences in degree of maturation during harvest, environmental fluctuations, genetics, location, soil type, irrigation conditions, year, and fertilizer application¹⁸.

Table (2): The phytate content of samples

	PA content (g/100g)
Peanut	1.73 ± 0.18 ^a
Whole wheat	1.55 ± 0.03
White wheat flour	0.42 ± 0.14

^a Results are means ± SD of samples each repeated three times

Effect of roasting on phytate contents:

The percentage of decreased PA contents for peanut roasted for 10, 20 and 30 min were between 3.64 - 5.78%, 12.18 - 22.6% and 14.25 - 25.76%, respectively [Table (3)]. The duration of roasting 20 min was sufficient to dispose of a large amount of phytic acid. These values are fairly close with published data where the percentage of decrease for phytic acid after 20 min of roasting was 12.76% by Ejigui et al¹¹ . The reduction in phytate contents may be due to partial damage to phytic acid at high heat.

Table (3): Percentage of decreased PA content during roasting of peanut

Roasting time (min)	Sample 1	Sample 2	Sample 3
10	5.78%	5.47%	3.64%
20	22.6%	14.18%	12.43%
30	25.76%	14.25%	14.9%

Effect of germination on phytate contents:

The percentage of decreased PA contents during germination of whole wheat were between 9.09 - 14.98% [Table (4)]. These values are fairly close with published data where the percentage of decrease for phytic acid after 3 days of germination was 17.86% by Azeke et al¹. The reduction in phytate contents may be due to increase the phytase activity in germination.

Table (4): Percentage of decreased PA content during germination of whole wheat

Samples	The percentage of decrease (%)
1	14.98
2	9.09
3	14.68

Effect of fermentation and baking on phytate contents:

The percentage of decreased PA contents for white wheat flour were between 15.22 - 37.56% for dough, 18.58 - 39.38% for dough with baking, 38.94 - 47.43% for dough with fermentation and 39.65 - 48.19% for dough with fermentation and baking [Table (5)]. The results indicate that knead the dough clearly reduce phytic acid content, this reduction may be due to fermentation during kneading. The fermentation has significant effect on the reduction of phytic acid may be due to increase the phytase activity in fermentation. The baking reduce phytic acid may be due to partial damage to phytic acid at high heat.

Table (5): Percentage of decreased PA content during fermentation and baking of white wheat flour

	Sample 1	Sample 2	Sample 3
D ^a	34.01%	15.22%	37.56%
DB	36.76%	18.58%	39.38%
DF	47.43%	38.94%	45.08%
DFB	48.16%	39.65%	48.19%

^aD: dough without fermentation and baking, DB: dough without fermentation but with baking, DF: dough without baking but with fermentation, DFB: dough with fermentation and baking.

These values agree with published data where the percentage of decrease for phytic acid after fermentation for 30 min at 30 ^◦C and baking for 30 min at 200 ^◦C was 50% by Garcia-Estepa et al⁴. The percentage of decrease after fermentation for 30 min and baking was 42% and after baking without fermentation was 13.5% by Qazi et al¹⁹. The percentage of decrease after fermentation for 30 min was 40% by Nayini et al³.

CONCLUSION:

As shown in this study, roasting, germination, fermentation and baking affected phytic acid contents in cereal grains. However, fermentation caused significant loss in phytic acid, while germination, roasting and baking caused less loss.

CONFLICT OF INTEREST:

The authors declare that there is not any conflict of interest related to this work.

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Received on 24.12.2018 Modified on 21.01.2019

Research J. Pharm. and Tech. 2019; 12(6): 2973-2976.

DOI: 10.5958/0974-360X.2019.00502.X