phytate reduction in oats during malting

Phytate Reduction in Oats during Malting

M. LARSSON and A.-S. SANDBERG

ABSTRACT TO reduce phytate in oat products and thereby increase mineral bioavailability, optimal conditions for phytate degradation in oats were investigated. The effects of malting and incubation on phytate reduction in oats were determined and compared with phytate degradation in wheat, rye and barley. Studies of incubation temperatures showed an optimum for phytate reduction in oats between 37-4O”C which differed from that in wheat (55°C). Malting of oats for 5 days at 11°C and subsequent incubation for 17 hr at 37-4O”C reduced phytate by 98%. Addition of malted rye also reduced the phytate content of oats to low levels.

Key Words: phytate, oats, wheat, barley, malting, phytase

INTRODUCTION CEREAL GRAINS are important sources of protein, minerals and vitamins. The bioavailability of these nutrients is affected by many complex and often interrelated factors (Betschart, 1988). Phytate, a compound normally present in large amounts in cereals, is suggested to be the major factor causing impaired absorption of several essential elements, such as zinc, iron, magnesium and calcium (Morris, 1986). Phytate concentration in cereals can be lowered by activation of the naturally occur- ring enzyme phytase, which hydrolyzes phytate to phosphate and myoinositolphosphates, thereby increasing mineral availability (Sandberg and Svanberg, 1991; Svanberg and Sand- berg, 1989; Nivert et al., 1985; Brune et al., 1992).

Bartnik and Szafranska (1987) found a substantial increase in phytase activity during germination of wheat, barley, rye and oats. They also confirmed earlier work (Peers, 1953; McCance and Widdowson, 1944; Mellanby, 1950) which in- dicated that different grains had different levels of phytase activity, with the highest being in rye, followed by wheat and barley, with the lowest in oats. The high content of phytate combined with low phytase activity and heat inactivation during treatment of all commercial oat products, suggests that mineral absorption of oat products is less than that of other cereals (Rossander-HulthCn et al., 1990; Sandstrijm et al., 1987; Sandberg and Larsson, 1991).

Our objective was to study whether it was possible to acti- vate oat phytase by malting and whether optimal temperature and pH for phytase activity in oats differed from those of rye, wheat and barley. Malting of wheat, rye, barley and oats followed by grinding and incubation were performed under different conditions. Addition of sprouted rye and barley as a potential source of phytase in oats was also investigated. The malted grains were intended for use as flour.

MATERIALS & METHODS Germination of oats, wheat, rye and barley

Oats, wheat, rye and barley were supplied by Kungsdrnen AB, Jima, Sweden. Prior to experiments the cereals were kept in paper bags at 0°C. Samples of each grain (50 g) were soaked with hulls in distilled water (500 mL) for 8 hr at room temperature (~23°C). Cer- eals were allowed to drain for 2 hr and the soaking was then repeated

Authors Larsson and Sandberg are affiliated with Chalmers Uni- versitv of Technoloav. Deot. of Food Science. P.O. Box 5401. S- 402 2b Gdteborg, S-iederk

for 1 hr. For germination, cereals were spread in plastic beakers with nylon screen in the bottom. Sprouts were grown in a water-bath to a length of about 10-15 mm for 3040 hr at 15°C. After sprouting, cereals were dried overnight at 37°C. Dried sprouts were ground to flour in a laboratory mill (Model Krups KM 75). Particle size was less than 0.50 mm as determined by sieving through a 0.50 mm screen. Sprouted flour samples (lg) of each grain were suspended in 10 mL distilled water and the pH of the mixture was adjusted to 4.5 by addition of 1M HCI using a Beckman +71 pH-meter. Two replicate samples (lg) were removed from each type of flour. Wheat, rye and barley samples were incubated in a waterbath at 55°C for 10, 20, 40, 60 and 120 min., and oat samples were incubated for l-20 hr. The influence of pH on phytate reduction in malted oats was studied by adjusting pH with 25% citric acid to pH-values of 3.3-5.1, and sus- pending sprouted oat flour in 10 mL O.lM citrate buffer, pH 5.3, instead of distilled water.

After different methods for improving phytate reduction in oats were tested, the final procedures used were: oats (cultivar Sang) were dehulled with a laboratory dehuller at Nyglrd Kvam, Vargen, Swe- den, and kept in a glass flask at 18°C prior to experiments. Dehulled oat grain (50g) were soaked in 500 mL distilled water for 7 hr at room temperature (~23°C). The soaked oats were spread in single layers on trays and dried in a laboratory incubator (Memmert Model BKE 30) at 30°C for 1 hr. Immediately after drying, a sample was removed for moisture determination. The oats were then covered with perfo- rated, damp paper towels to minimize moisture loss and germination was begun. During the germination period, the oats were moistened with distilled water 2-3 times during the day. Incubator was held at 11°C. The oats were allowed to germinate for 5-7 days (sprouts ap- prox. lo-15 mm) and dried overnight at 30°C. The dried grains were ground to flour, which was frozen and freeze-dried prior to incubation.

Sprouted oat flour was incubated in three separate experiments. In the first (I) lg sprouted oat flour was suspended in 10 mL distilled water and the pH of the mixture was adjusted to 4.5 by addition of 1M HCl. The samples were incubated for 4 hr at different temperatures (29”, 32”, 35”, 37”, and 48°C). In the next experiment (II) 0.2g sprouted rye or barley flour was added to lg malted oat flour and suspended in 10 mL distilled water. pH was adjusted with 1M HCl to 4.5 and the samples were incubated for 4 hr at 35, 37 or 55°C. In the third experiment (III) lg sprouted oat flour was suspended in 10 mL distilled water and incubated overnight (17 hr) at 20, 37, 38, 39, 40 and 41°C without pH adjustment. pH measurements were made at the start and end of .incubations. tie or more replicate samples were taken in each separate test.

Moisture determination

Dry matter was determined 9;~ drying for 17 hr at 105°C. All values were calculated on a dry matter basis.

Analytical procedures

Phytate determinations. Incubated samples. To stop phytate degradation 30 mL 0.67M HCI was added at the end of incubation. The samples were then extracted for 3 hr. The inositol phosphates were separated from the crude extract by ion exchange chromatography and determined by ion pair Cl8 reverse phase HPLC using formic acid/ methanol and tetrabutylammoniumhydroxide in the mobile phase. The amounts of inositol tri-, tetra-, penta- and hexaphosphates were determined by HPLC according to Sandberg and Ahderinne (1986) and Sandberg et al. (1987). The analysis was performed using a HPLC pump (Waters Model 510, Waters Ass. Inc. A) equipped with a Cl8 Chromasil (5 pm) column 2 mm i.d., and the inositol phosphates were detected by refractive index (ERG7510 RI-detector, Erma Op- tical Works Ltd., Japan). Retention times and peak areas were mea-

994-JOURNAL OF FOOD SCIENCE-Volume 57, No. 4, 1992

Sample

Oats

Wheat

Rve

Barley

Table I-lnositol phosphates” Ipmollg dry weight) in germinated (3040 hr, 1SC) and ground cereals incubated at 55°C and pH 4.5 Time of inc IP$

Phytate IP, IP5 IPS reduction’

(min) bmoVd % -c 0.25 0.04 k 17.25dzo.91

0 0.17+0.01 0.36kO.01 14.54kO.47 16 60 0.3420.05 1.58*0.11 2.54-cO.12 8.07 f 0.23 53

240 0.31 kO.01 2.04kO.04 2.70 eO.05 7.34eO.20 57 480 0.28 2 0.05 2.66 f 0.06 2.54-cO.01 6.4O-cO.13 63

1200 0.71 kO.01 3.08 f 0.05 2.58 f 0.08 6.14k0.18 64 -c

0.26;0.05 0.55kO.04 10.28-tO.67

0 0.63 f 0.06 10.08kO.42 2 IO 2.4820.63 3.04dzO.21 0.92*0.44 2.49kO.57 76 20 t+ tr 0.07*0.04 1.69k0.13 84 40 tr tr 0.06kO.01 1.441ro.30 86 60 tr tr 0.03 * 0.04 0.87kO.16 92

120 tr tr 0.02-+0.01 0.52 f 0.08 95 -c

o.10t~o.14 0.12:0.01 0.68*0.11 9.31 kO.05

0 0.63zkO.01 9.1620.12 2 10 0.39 f 0.05 0.10~0.01 0.18kO.01 2.58*0.07 72 20 0.06 +- 0.04 0.04~0.01 0.12*0.01 1.56 f 0.02 83

40 0.02 z!c 0.03 0.01+0.02 O.OE*O.Ol 0.97~0.10 60 0.04*0.03 0.01 kO.02 0.04~0.01 0.74~0.03 ii 120 tr tr 0.02 rO.O1 0.49 20.02 95 -c -

0 0.20T0.28 0.48 f 0.08 12.13r0.60 0.33eO.06 6.0520.17 50

10 1.25 f 0.68 3.52 + 0.33 1.59t0.10 2.45 k 0.32 80 20 3.26~kO.32 3.8920.31 0.36 + 0.03 0.57” 0.06 40 4.93 f 0.09 1.66*0.01 0.05-c0.01 0.15+0.06 ii 60 3.39 * 0.01 0.29~0.01 tr 0.06 + 0.01 99

120 tr tr tr tr a Values represent mean + SD of at least two replicates. a IPJ to IP, - inositol containing three to six phosphates par inositot residue. C Untreated grains. dtr = tracea. 0 Percentage phytate reduction (IPs) calculated on initial content in untreated samples.

sured by a laboratory data system, HP 1000 (Hewlett Packard). Injections were made with a 20 pL loop.

Sodium phytate (BDH Chemical, England) was used as the external standard and 20 ~.LL injections of standard solutions containing 0.05, 0.1, 0.2, 0.5 and 1.0 ~mol/ml of phytate were prepared. To achieve a linear standard curve several injections of a freshly prepared standard containing 5 umol/ml of phytate were made before starting analysis. Correction factors for differences in detector response of inositol hexa-, penta-, tetra- and triphosphates were calculated as described by Sandberg and Ahderinne (1986). The precision of the method was tested by repeating the extraction and analysis of the same batch of wheat bran in 12 replicate samples. The mean 2 SD of wheat bran samples was 46.9 c 1.5 umol/g (dry basis). The same procedure was applied to four samples of rye (whole meal flour) and four samples of phytase deactivated oats (whole meal flour). Mean values ? SD were 10.3 -+ 0.2 and 9.9 2 0.35 pmol/g inositol hexaphosphates (dry basis), respectively. The accuracy of the HPLC method was estimated from these results to be about -C 3%.

To determine phytate content in oats before and after germination, control samples of ungerminated, milled cereals and germinated but not incubated cereals (0.5 g) were analyzed using the same procedure as for incubated samples.

Determination of minerals in oats. Samples of freeze-dried, malted oats and untreated oats were analyzed in duplicate for contents of iron, zinc, magnesium and calcium. Fe, Zn and Mg were determined after dry-ashing and Ca after wet-ashing. The minerals were determined vs blanks in an atomic absorption spectrophotometer (Perkin Elmer 360).

RESULTS

PHYTATE REDUCTION in oats, wheat, rye and barley during malting at 15°C for 30-40 hr was determined to be 16, 2, 2 and 50% respectively. Table 1 gives the content of inositol phosphates in germinated and ground cereals, incubated at 55°C and pH 4.5. Under those conditions the phytate reduction in oats was much lower compared with other cereals. 36% of the phytate content in oats remained present even after 20 hr incubation. In germinated wheat, rye and barley the initial phytate content (inositol hexaphosphate) was reduced after 10 min incubation by 76, 72 and 80%, respectively. During further incubation the phytate content in the sprouted flour from wheat,

rye and barley continued to decrease and within 2 hr the reduction was 95% or more. The greatest phytate reduction occurred in germinated barley, where only traces were found after 2 hr incubation. The effect of pH on phytate reduction in sprouted and incubated oat flour was tested at the pH interval 3.3-5.1 after 4 hr incubation at 55°C (pH adjusted with citric acid). In all cases, phytate degradation was low (about 25%). No significant differences were observed between different pH values.

Incubation at 55°C for 4 hr of the sprouted oat flour, suspended in citrate buffer, did not further increase phytate reduction.

In Experiment I phytate reduction in oats germinated for 5 days at 11°C was temperature dependent. The greatest reduction (85% of the initial value) was achieved by incubating the samples at 37°C and pH 4.5 for 4 hr (Table 2). Further germination (for 7 days) did not enhance phytate reduction. In this experiment the moisture content of soaked and dried (1 hr at 30°C) oats was about 23% at start of germination. The phytate content in ungerminated oats was reduced by 29% after incubation at 37°C and pH 4.5 for 4 hr.

In Experiment II additions of sprouted rye and barley flour to sprouted oat flour further increased phytate hydrolysis (Ta- ble 2, Exp. II). The greatest reduction (93%) occurred in germinated oats incubated at 35 or 37°C with addition of sprouted rye.

In Experiment III incubation of ground samples at room temperature for 17 hr resulted in degradation of 94% of the phytate content in oats germinated for 5 days at 11°C (Table 2, Exp. III). During the first hour of incubation the pH dropped from about 6 to 5.0. Soaking for 17 hr at 37 to 40°C reduced phytate content by 97-98%. At the end of incubation the pH of the samples was about 3.5.

Determination of minerals

The contents in ungerminated oat flour were iron 70.7~0.20 mmol/lOOg, zinc, 56.3 2 0.48 p,mol/lOOg, magnesium 5.2 4 0.00 mmol/ lOOg and calcium 1.4 & 0.00mmo1/100g. The

Volume 57, No. 4, 1992-JOURNAL OF FOOD SCIENCE-995

PHYTATE REDUCTION IN OATS.. .

Table Z-lnositolphosphates’ (co//g dry weight) in ground oats after germination and incubation Time of

germ Time of Temp 11°C inc of inc IPJb IP4 lb IP6

(Ws) (hr) ("Cl (wmll9) Exp.1

: 4 0 37 0.83:0.04 1.16;0.02 0.18-+0.01 2.10+0.04 17.3OkO.34 12.24~0.74

5 0 0.12kO.17 0.16+0.01 0.35kO.02 14.OOzkO.81 5 4 29 3.96*0.10 3.0920.28 0.9OzO.16 3.07kO.31 5 4 32 3.48k1.48 3.38r0.70 0.74+0.02 2.76-cO.04 5 4 35 4.18+0.01 3.01+0.20 0.86r0.10 2.79kO.08 5 4 37 3.48k0.93 2.90r0.02 0.73+- 0.01 2.53kO.02 7 0 0.39r0.34 0.20~0.03 15.6420.52 7 4 37 3.79kO.25 2.2OkO.30 0.63kO.22 3.42eO.34 7 4 48 1.43kO.10 2.69-cO.40 3.3OzkO.06 8.03?0.07

Exp. II 0 4d 55 0.08~~0.07 0.08r0.01 0.09*0.04 3.22kO.03 5 ;: 35 0.22zko.30 0.09 e 0.01 0.07r0.01 1.28 t 0.03 5 37 0.22kO.01 0.09 f 0.01 0.07~0.01 1.24kO.06 5 4d 55 tr 0.07*0.02 0.08kO.01 2.06-cO.15 5 4' 37 5.31kO.11 3.4520.10 0.50~0.01 1.71~0.04 5 40 55 4.24k1.41 7.5620.38 1.37kO.02 3.57eo.01

Exp. Ill 5 17 20 0.72kO.07 0.51-co.04 0.14-cO.02 0.95-co.04

5 17 37 0.09~0.02 0.05-+0.01 0.03-+0.01 0.46?0.04 5 17 38 0.62kO.22 0.14-co.03 0.07~0.04 0.29kO.13 5 17 39 3.31k0.52 0.99 kO.18 0.20*0.07 0.27+0.06 5 17 40 3.14-co.44 2.24-cO.28 0.41~0.04 0.38kO.10 5 17 41 5.29-to.24 4.07kO.41 1.23-r-0.15 0.82+0.11

a Values represent mean + SD of at least two replicates. b IPs to IPB = inositol containing three to six phosphates per inositol residue. C Percentage phytate reduction (IPs) calculated on initial content in untreated sample. d With addition of 0.2Og sprouted rye. 0 With addition of 0.2Og sprouted barley.

Phytate red'

%

29 19 82

i: 85 10 80 54

81 93 93 87 90 79

94

iii 98 98 95

corresponding amounts in oats germinated for 5 days at 11°C were 77.22 0.81 mmol/lOOg 58.42 0.38 pmol/lOOg, 5.4+ 0.01 mmol/lOOg and 1.5 2 0.08 mmol/lOOg.

DISCUSSION

WE FOUND the optimum temperature for phytate reduction in malted oats (37-4o”C) differed from that of wheat and rye (55°C). Malting of oats for 5-7 days at 11°C and subsequent soaking of ground oats at pH 5 (pH adjusted with HCl) for 4 hr at 37°C decreased the inositol hexaphosphate content by 80-85%. For comparison we found that around 29% of the initial inositol hexaphosphate content was hydrolysed in untreated and ground oats during incubation under the same conditions. In oats malted 40 hr at 15°C and incubated at 55°C for 20 hr the phytate content was reduced by 64%. The results may indicate that some changes, involving either production or activation of phytase, take place during the germination period and that the temperature and duration of the germination are likely very important factors in improving phytase activity in oats. A similar observation was made by Albaum and Um- brecht (1943), who studied phosphorus transformations during development of the oat embryo. They found that phytase activity appeared to increase with increased germination time and that phytate hydrolysis did not begin to occur rapidly until 72 hr after germination. By germination for 120 hr followed by incubation at 37°C for 6 hr at pH 6.3, they reached a phytate reduction of 79%, to which our results are comparable.

We found that it was possible to almost completely reduce the phytate content of oats (to < 0.5 p,mol/g) by germination at 11°C for 5 days followed by incubation at 37 to 40°C for 17 hr. An unexpected finding was that when the malted oats were incubated without addition of HCl, the acidity was reduced to optimal pH conditions, resulting in an almost complete removal of initial phytate content. Since it has been found that even small amounts of phytate have a marked negative effect on iron availability (Sandberg et al. 1989; Hallberg et al. 1989), this low level is desirable to greatly improve the bioavailability of iron from oats. The content of iron, zinc,

calcium and magnesium in the malted oat flour was unaffected by our procedure. (A small increase of the percentage of total mineral content was found, which probably could be explained by losses of dry matter in sprouted oats.)

In our study, sprouted barley had the most effective phytate decomposition of the 4 cereals. We found that the phytate content in sprouted and ground barley became almost completely degraded during the first hour of incubation at 55°C. This agreed with a recent report by Frolich (1990), who studied the breakdown of phytate in rye, wheat, barley and oats during incubation in water at 37°C for O-22 hr. The most rapid hydrolysis was reported to occur in barley, where all phytate present was broken down after 4 hr incubation. For the other cereals, rye, wheat and oats (not heat-treated), incubation for 4 hr under the same conditions led to phytate reduction of 82, 71 and 34%, respectively. We previously found a similar value for oat flour (33%), but a complete degradation in rye flour when soaking for 4 hr at pH 5, 55°C (Sandberg and Svanberg 1991). Our corresponding values after malting and incubation at 55°C for 1 hr were 92, 92 and 53%, respectively. This was, however, in contrast to the studies of Bartnik and Szafranska (1987), who compared changes in phytate content and phytase activity during germination of rye, wheat, barley and oats for 6-96 hr at 20°C. They reported that the phytase activity in germinated rye was higher than that in germinated barley (di- rect comparison with our results is, however, difficult, since conditions were dissimilar).

The amount of phytate in barley was reduced by 50%. In malted grains of oats, wheat and barley the phytate content was reduced only slightly or not at all. When the malted cereals were ground and soaked at optimal conditions for wheat phytase (pH 4.5-5.5, 55°C) there was an almost complete degradation of phytate after 2 hr except for oats, which under the conditions studied had 1.0~ phytase activity (as measured by phytate reduction). Under these conditions adjustments of pH with citric acid and citrate buffer to achieve a more efficient phytate hydrolysis during the subsequent incubations of malted oats did not cause noticeable changes in phytate levels. Pre- vious studies performed on phytase activity in oats are scarce

996-JOURNAL OF FOOD SCIENCE-Volume 57, No. 4, 1992

and the results are conflicting, either indicating no (Hoff-Jor- gensen et al. 1946) or low to medium activity (Bartnik and Szafranska 1987; Lockhart and Hurt 1986). Since it has been reported that phytase activity could be inhibited by high substrate concentration (Beal and Mehta, 1985; Dagher et al., 1987; Peers, 1953), it must be taken into account that the speed and degree of phytate hydrolysis might depend on how high the phytate content is from the start. We found a considerably higher initial phytate content in oats compared with other cereals. This could, however, partly be explained by the fact that the husk, which was removed in the preparation, forms about 30% by weight of the grain. Since the husk contains no phytate, the percentage of phytate in the dehusked grain would rise correspondingly.

Addition of sprouted rye or barley to oats before soaking also resulted in phytate reduction to low levels. Phytase activity as measured by phytate degradation increased markedly during incubation at 35, 37 and 55°C and the greatest activity was observed in samples with addition of sprouted rye, incubated at 35 or 37°C for 4 hr. We also found that addition of sprouted rye to ungerminated and ground oats reduced the phytate content by 81% (Table 2, Exp. II). Thus, utilization of germinated rye as a potential source of phytase might be an- other way to reduce phytate in oats and oat products.

CONCLUSIONS MALTING of cereals followed by soaking at optimal conditions could be used to obtain flour with a high mineral availability, as phytate is considered to interfere with absorption of minerals. Such flour could be a significant source of minerals when used e.g. in production of infant formulas and breakfast cereals.

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This work wan supported by grants from the Cerealia Foundation R&D, Stockholm, Sweden. and the Swedish Nutrition Foundation.

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phytate reduction in oats during malting

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