Die Nahrung 34 (1990) 1,99- 101
, Central Institute of Nutrition in Potsdam-Rehbriicke (Direktor: Prof. Dr. H. SCHMANDKE), Academy of Sciences of the GDR
Modified method of phytic acid analysis in rapeseed flour (Short communication)
R. MOTHES,K. D. SCHWENKE and S. GROSSMANN
Introduction A wide-spread method for the analysis of phytic acid is based on the determination of phosphorus in ironphytic acid complexes after precipitation of phytic acid from acid extracts by ferric ions [l]. The extraction of phytic acid was performed mostly by means of hydrochloric acid containing sodium sulphate. Trichloroacetic acid (TCA) was also recommended as an extractant. The latter has the advantage over HCI (even HCl with Na2S04) of preventing the extraction of some soluble proteins which may contain contaminating phosphate and be precipitated or coprecipitated with ferric ions in dilute acid [2]. The concentratioii of TCA was widely varied to reach complete extraction. Conditions producing a pH approaching zero were also used [3]. As the minimum solubility of iron phytate was found in the range of pH 2.5-8.0 [4] the question arises whether a quantitative precipitation of the complex takes place at lower pH. The present paper is concerned with the conditions of maximum precipitation of iron phytate from a TCA-containing extract.
Materials and methods 1 g rapeseed flour (Brassicu nupus L., vur. Murinus) was extracted with 100 ml 5% TCA during 2 h at temperature. After centrifugation (4200 x g, 20 min) the extract was transferred into a 250 ml volumetric flask and the volume refilled with distilled water. To an aliquot of 25 ml of this extract 20 ml of iron(II1) chloride (0.4% in 0.6 % HCI) solution and in the modified version additionally some ml of sodium acetate solution (2.5 N) were added and the mixture was heated for 30 min on a boiling water bath. After cooling and centrifugation the clear supernatant was decanted and discarded. After adding 10 ml conc. sulphuric acid the precipitate was transferred with a small amount of water into an ERLENMEYER flask and evaporated on a heating plate until fumes of SO3 appeared. By dropwise adding 1 ml of hydrogen peroxide (30%) a decolourization took place. The mixture was then heated again until the development of SO,. If necessary the addition of hydrogen peroxide can be repeated until the complete de1 ml H,SO,) was treated analogously. colourization. The blank (distilledwater After mineralization the sample was heated for 15 min in a boiling wather bath to destroy pyrophosphate. After cooling the sample was transferred into a 50 ml volumetric flask and the volume filled up with distilled water. To determine the total phosphorus 100 mg of the flour were suspended in water and mineralized analogously. Depending upon the phytic acid content 2 to 10 ml of the solution were neutralized with 5.4 N NaOH. 20 g Na2S04 . 7 H 2 0 and 150 g Then 2.5 ml of reduction solution (2 g monomethyl-p-aminophenolsulphate, NaHSO, per 1) and 5 ml ammonium molybdate ( 5 % in 5 N H2S04)were added. Exactly 10 min after addition of the latter 10 ml of 2.5 N sodium acetate solution were added. The samples were filled up to 25 ml with distilled water. 40 min after adding the molybdate reagent the absorption at 720 nm was measured against the blank. A solution of NaH2P04was used for calibration.
+
Results Table 1 shows the results of phytic acid determination in three different rapeseed meals using the precipitation of iron phytate from the TCA extract both in the presence and in the absence of sodium acetate. 7*
100
Die Nahrung 34 (1990) 1
Table 1 Content of phytic acid phosphorus* in three different rapeseed flours determined both in the presence and absence of sodium acetate (Naac) Sample No 1 2 3
*
Total P [mg/gI
Phytic acid phosphorus [mg/g]
+ 0.5 ml Naac
without Naac
11.3 f 0.1 7.6 f 0.1 9.5 0.2 14.9 f 0.1 10.4 f 0.1 6.3 f 0.2 each extract was analyzed in triplicate
9.4 f 0.1 12.1 f 0.1 8.5 f 0.1
*
/
f
/
/"
/-
/
/
/-
/'
d/ 0
Fig. 1 Influence of the sodium acetate concentration on the change of pH and the precipitated amount of iron phytate (calculated as the amount of phytic acid phosphorus determined)
0.1 a2 0.3 0.4 0.5 concentmtion of sodium acetate lmmol /I 'I
Precipitation in the presence of sodium acetate gave a content of phytic acid phosphorus corresponding to 81-83 % of the total phosphorus. These values correspond well to those obtained by means of enzymatic hydrolysis with phytase [5].Precipitation in the absence of sodium acetate resulted, however, in lower contents of phytic phosphorus corresponding to 60-67 % of total phosphorus. As Fig. 1 shows the precipitated amount of iron phytate corresponding to higher phytic acid content in the flour increases with increasing addition of sodium acetate until a total concentration of 0.25 mM in the sample solution. Further increase of sodium acetate concentration did not influence the amount of phytic acid determined. The pH of the sodium acetate-free solution was 0.7. It increases to 0.9 when the sodium acetate concentration was increased to 0.25 mM. Enhancement of the salt concentration to 0.5 m M resulted in an increase of pH to 3.5.
Conclusion The presence of sodium acetate improves the precipitation of the iron phytate complex from TCA containing extracts. Maximum amount of the complex was precipitated at a sodium acetate concentration of 0.250.50 mmol/l in the pH-range of 0.9-3.5.
WissenschaftlicherKurzbericht
101
References
[I]OBERLEAS, D., in: Methods of Biochem. Analysis, Vol. 20, p. 87.John Wiley & Sons, Inc., New York, London, Sydney, Toronto 1971. [2]OBERLEAS, D.,in: Methods of Biochem. Analysis, Vol. 20,p. 95.John Wiley & Sons, Inc., New York, London, Sydney, Toronto 1971. 13) FONTAINE, T. D., W. A. PONDSand W. G. IRVING, J. Biol. Chem. 164, 487 (1946). [4]JACKMAN, R . k . , and C. A. BLACK,Soil. Sci. 72, 179 (1951). [5]UPPSTROM, B., and S. SVENSSON, J. Sci. Food Agric. 31,651 (1980). Dr. R. M o m , Prof. Dr. K. D. SCHWBNKE and SIEGLINDE GROSSMANN, Central Institute of Nutrition, Arthur-Scheunert-Allee 1 14- 116,Bergholz-Rehbriicke, DDR- 1505 Received August 17, 1989
, Central Institute of Nutrition in Potsdam-Rehbriicke (Direktor: Prof. Dr. H. SCHMANDKE), Academy of Sciences of the GDR
Modified method of phytic acid analysis in rapeseed flour (Short communication)
R. MOTHES,K. D. SCHWENKE and S. GROSSMANN
Introduction A wide-spread method for the analysis of phytic acid is based on the determination of phosphorus in ironphytic acid complexes after precipitation of phytic acid from acid extracts by ferric ions [l]. The extraction of phytic acid was performed mostly by means of hydrochloric acid containing sodium sulphate. Trichloroacetic acid (TCA) was also recommended as an extractant. The latter has the advantage over HCI (even HCl with Na2S04) of preventing the extraction of some soluble proteins which may contain contaminating phosphate and be precipitated or coprecipitated with ferric ions in dilute acid [2]. The concentratioii of TCA was widely varied to reach complete extraction. Conditions producing a pH approaching zero were also used [3]. As the minimum solubility of iron phytate was found in the range of pH 2.5-8.0 [4] the question arises whether a quantitative precipitation of the complex takes place at lower pH. The present paper is concerned with the conditions of maximum precipitation of iron phytate from a TCA-containing extract.
Materials and methods 1 g rapeseed flour (Brassicu nupus L., vur. Murinus) was extracted with 100 ml 5% TCA during 2 h at temperature. After centrifugation (4200 x g, 20 min) the extract was transferred into a 250 ml volumetric flask and the volume refilled with distilled water. To an aliquot of 25 ml of this extract 20 ml of iron(II1) chloride (0.4% in 0.6 % HCI) solution and in the modified version additionally some ml of sodium acetate solution (2.5 N) were added and the mixture was heated for 30 min on a boiling water bath. After cooling and centrifugation the clear supernatant was decanted and discarded. After adding 10 ml conc. sulphuric acid the precipitate was transferred with a small amount of water into an ERLENMEYER flask and evaporated on a heating plate until fumes of SO3 appeared. By dropwise adding 1 ml of hydrogen peroxide (30%) a decolourization took place. The mixture was then heated again until the development of SO,. If necessary the addition of hydrogen peroxide can be repeated until the complete de1 ml H,SO,) was treated analogously. colourization. The blank (distilledwater After mineralization the sample was heated for 15 min in a boiling wather bath to destroy pyrophosphate. After cooling the sample was transferred into a 50 ml volumetric flask and the volume filled up with distilled water. To determine the total phosphorus 100 mg of the flour were suspended in water and mineralized analogously. Depending upon the phytic acid content 2 to 10 ml of the solution were neutralized with 5.4 N NaOH. 20 g Na2S04 . 7 H 2 0 and 150 g Then 2.5 ml of reduction solution (2 g monomethyl-p-aminophenolsulphate, NaHSO, per 1) and 5 ml ammonium molybdate ( 5 % in 5 N H2S04)were added. Exactly 10 min after addition of the latter 10 ml of 2.5 N sodium acetate solution were added. The samples were filled up to 25 ml with distilled water. 40 min after adding the molybdate reagent the absorption at 720 nm was measured against the blank. A solution of NaH2P04was used for calibration.
+
Results Table 1 shows the results of phytic acid determination in three different rapeseed meals using the precipitation of iron phytate from the TCA extract both in the presence and in the absence of sodium acetate. 7*
100
Die Nahrung 34 (1990) 1
Table 1 Content of phytic acid phosphorus* in three different rapeseed flours determined both in the presence and absence of sodium acetate (Naac) Sample No 1 2 3
*
Total P [mg/gI
Phytic acid phosphorus [mg/g]
+ 0.5 ml Naac
without Naac
11.3 f 0.1 7.6 f 0.1 9.5 0.2 14.9 f 0.1 10.4 f 0.1 6.3 f 0.2 each extract was analyzed in triplicate
9.4 f 0.1 12.1 f 0.1 8.5 f 0.1
*
/
f
/
/"
/-
/
/
/-
/'
d/ 0
Fig. 1 Influence of the sodium acetate concentration on the change of pH and the precipitated amount of iron phytate (calculated as the amount of phytic acid phosphorus determined)
0.1 a2 0.3 0.4 0.5 concentmtion of sodium acetate lmmol /I 'I
Precipitation in the presence of sodium acetate gave a content of phytic acid phosphorus corresponding to 81-83 % of the total phosphorus. These values correspond well to those obtained by means of enzymatic hydrolysis with phytase [5].Precipitation in the absence of sodium acetate resulted, however, in lower contents of phytic phosphorus corresponding to 60-67 % of total phosphorus. As Fig. 1 shows the precipitated amount of iron phytate corresponding to higher phytic acid content in the flour increases with increasing addition of sodium acetate until a total concentration of 0.25 mM in the sample solution. Further increase of sodium acetate concentration did not influence the amount of phytic acid determined. The pH of the sodium acetate-free solution was 0.7. It increases to 0.9 when the sodium acetate concentration was increased to 0.25 mM. Enhancement of the salt concentration to 0.5 m M resulted in an increase of pH to 3.5.
Conclusion The presence of sodium acetate improves the precipitation of the iron phytate complex from TCA containing extracts. Maximum amount of the complex was precipitated at a sodium acetate concentration of 0.250.50 mmol/l in the pH-range of 0.9-3.5.
WissenschaftlicherKurzbericht
101
References
[I]OBERLEAS, D., in: Methods of Biochem. Analysis, Vol. 20, p. 87.John Wiley & Sons, Inc., New York, London, Sydney, Toronto 1971. [2]OBERLEAS, D.,in: Methods of Biochem. Analysis, Vol. 20,p. 95.John Wiley & Sons, Inc., New York, London, Sydney, Toronto 1971. 13) FONTAINE, T. D., W. A. PONDSand W. G. IRVING, J. Biol. Chem. 164, 487 (1946). [4]JACKMAN, R . k . , and C. A. BLACK,Soil. Sci. 72, 179 (1951). [5]UPPSTROM, B., and S. SVENSSON, J. Sci. Food Agric. 31,651 (1980). Dr. R. M o m , Prof. Dr. K. D. SCHWBNKE and SIEGLINDE GROSSMANN, Central Institute of Nutrition, Arthur-Scheunert-Allee 1 14- 116,Bergholz-Rehbriicke, DDR- 1505 Received August 17, 1989
