Effect of meal composition and phytate content on zinc absorption in humans from an extruded bran product

Journal oj Cereal Science 10 (1989) 189-197

Effect of Meal Composition and Phytate Content on Zinc Absorption in Humans from an Extruded Bran Product BARBRO KIVISTO*t, AKE CEDERBLADt, LENA DAVIDSSON*, ANN-SOFI SANDBERG§ and BRITTMARIE SANDSTROM~ University of Gothenburg, Departments of * Clinical Nutrition and t Radiation Physics, Annedalsklinikerna S-413 45 GOteborg, Sweden, § Chalmers University of Technology, Department of Food Science, Goteborg, Sweden and the ~ Royal Veterinary and Agricultural University, Research Department of Human Nutrition, Fredriksberg C, Denmark Received 7 April 1989

The effect of extrusion cooking of a high fibre cereal product on zinc absorption was studied using radioisotopic labelling of single meals and measurement of whole- body retention. Thirty-three subjects participated in the study. Three extruded products were tested containing 10% gluten, 20 or 30 % wheat bran and 60 or 70 % starch and compared to the corresponding non-extruded ingredients. One additional product was extruded after reduction of the phytate content. The test products were served with a cooked meal or with milk as the main ingredient of a breakfast. When served with a cooked high protein meal there was no difference in Zn absorption from an extruded product or corresponding raw materials, 36·6 ±2·0 % and 31-8 ±3·1 % respectively. Zn absorption from a breakfast including I SO g of an extruded product was only 6·2 ±0·6 %. However, Zn absorption was greatly improved when the extrusion was performed after reduction of the phytate content.

Introduction Extrusion cooking is a widely used food processing technique. It is normally a high temperature-short time (HTST) process using high shearing forces at elevated pressure. It is used for a wide variety of foods such as vegetable proteins, breakfast cereals, weaning foods, crisp bread, snacks and sweets. The nutritional implications of extrusion cooking have been poorly investigated, most concern has been with protein digestibility and starch availabilityl-3 and the implications of the formation of amylose-lipid cornplexes 4 . Very little is known of the effect of extrusion cooking on mineral availability. Lykken et al. 5 found reduced absorption of zinc from a browned corn product (cornflakes) compared to that from an unbrowned product (corn grits) and attributed Abbreviations used: HPLC = high performance liquid chromatography. t To whom correspondence should be addressed.

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this to heating and toasting reaction products formed during processing of the cornflakes. Fairweather-Tait et al. 6 found no effect of extruding maize or potato on fractional iron absorption in rats. In a study using ileostomates (subjects who for medical reasons have their small intestine leading to an opening on the abdomen where the visceral contents are poured out into a changeable bag), we have recently found a lower apparent absorption of zinc, magnesium and phosphorus from a diet which included an extruded high fibre cereal product compared to that from a diet including the non-extruded ingredients 7 • We also found a lower digestibility in the small intestine of the phytate from the extruded product compared to that from the ingredients, which could imply a lower availability of minerals 8 • It has been shown that phytate (inositol hexaphosphate) is partially degraded to inositol penta- and tetraphosphates during extrusion cooking 9 • These compounds may have other effects on mineral absorption than phytate. L6nnerdal et a/. lO found hexa- and pentaphosphates of inositol to reduce the uptake of Zn and ea in rats but no such effect for tetra- and triphosphates of inositol. To investigate further the effect of extrusion cooking on zinc absorption, we have in the present study used a more sensitive method than the balance method, i.e. radioisotopic labelling with 65Zn of single meals containing an extruded high fibre product or its ingredients and measurement of the whole-body retention of the radionuclide l l . An improved zinc absorption has earlier been observed from wheat bran after reduction of the phytate content by leavening 12 • In order to separate the effect of extrusion cooking and ofphytate content, zinc absorption was also measured from one test product which was extruded after reduction of the phytate content.

Subjects

Experimental

Twenty females and 13 males between 21 and 42 years of age (median 24 years) volunteered for the study. Sixteen subjects participated twice. They were all apparently healthy, non-pregnant, without kn~wn gastrointestinal disorders and had normal serum Zn levels of 11·6-18'6 (mean U9) J.lmoJjI. The subjects were given written and oral information about the aim and procedure of the study.

Zn absorption measurements Zn absorption was determined using the radionuclide technique described by Arvidsson et al. ll Each extrinsically labelled meal was measured in a whole-body counter. Each subject's background radioactivity was measured before intake of the labelled test meal. The subjects were randomly allocated to the meals. Test meals nos. 1 and 2 were served as lunch 3-4 h after intake of an unlabelled breakfast consisting of milk, bread, butter and tea or coffee. No food or drink was allowed 12 h before breakfast or between breakfast and lunch. Test meals nos. 3,4 and 5 were served after an overnight fast. Neither food nor drink was allowed for 3 h after intake of the test meal. The whole-body retention of the radioisotope was measured 10-14 d after intake of the meal to allow excretion of the unabsorbed fraction. Allowance was made for the excretion of initially absorbed isotope during the time between intake and retention measurement based on the mean rate of excretion of an intravenously administered dose of 65Zn in a similar group of subjects l l . When the subjects participated the second time, allowance was also made for the excretion of the residual radioactivity from the first meal.

EXTRUSION COOKING AND ZINC ABSORPTION

191

Extrusion cooking and conditions Three extruded products were tested, see Table I for composition and extrusion conditions. The extrusion was perf0rmed in a Creusot-Loire BC extruder (Firminy, France) with co-rotating double screw with the following configuration: transport, low pressure, medium-pressure, highpressure and reverse-screw elements. No external heat was transferred to the barrel or the screws during extrusion. Instead, the barrel was cooled with a specially-designed air-cooling device. Mass temperature and pressure were measured with a Dynisco probe in the compression chamber just before the dies. In product A and B the bran was extruded in its untreated state while in product C it was extruded after reduction of the phytate content through incubating the bran with an equal amount of citrate buffer (pH 3'0) at 55°C for 14,5 h. The final pH was 5·0 which is optimal for wheat phytase 13 •

Meals for Zn absorption studies All food was bought in bulk and prepared in advance of the study. The extruded product resembled crispbread and was served as such. The lunch meals (nos. 1 and 2) consisted of fried fillet of plaice (150 g), rice (40 g uncooked), and extruded product A (27 g) or the corresponding amounts of bran, starch and gluten. The rice was cooked in the oven (200°C) with deionized water (100 g) for 40 min, gluten and starch were added the last 5 min to give a palatable consistency. The meals were heated in an oven before serving. The bran was added just before serving. Meal no. 3 consisted of fermented milk (200 g)*, one roll (containing 20 g wheat flour), butter (5 g) and extruded product A (33 g; 30 % bran, 60 % starch and 10 % gluten). Meals nos. 4 and 5 consisted of fermented milk (200 g)* and extruded product B or C (150 g; 20 % bran, 70 % starch and 10 % gluten) made of raw bran or bran with reduced phytate content. Deionized water (200 g) was also served with all meals. Each meal was extrinsically labelled with 66Zn (0'02 MBq) by dripping almost carrier-free 66ZnCI 2 solution (0,8 MBq/l-lmol Zn; Amersham International, Amersham, Bucks) into the extruded product, to the ingredients or to the bran 15-30 min before serving.

Food analyses Portions of the freeze-dried food were analysed in duplicate for their contents of Zn, Fe, Ca, Mg, P, N, phytic acid and dietary fibre. The extruded product and the corresponding raw material were analysed separately. All glassware was washed in 2·5 M HCI and rinsed in deionized water before use. Zn and Fe were determined by atomic absorption spectrophotometry (Perkin Elmer Model 360), after dry-ashing at 450°C. Ca and Mg were determined by atomic absorption spectrophotometry after wet-ashing (290-300 °C, 15 min) in cone H 2S0 4 and hydrogen peroxide and after addition of lanthanum oxide. The same digest was used to determine P according to Fiske and Subbarow 14 • Reference standards for Zn, Fe, Ca and Mg were prepared from Titrisol® (Mercks). Reference standard materials for Zn and Fe with concentrations representative of those found in the diet were run simultaneously and fell within the certified range [Orchard Leaves SRM 1571 and Bovine Liver SRM 1577 (a), National Bureau of Standards, U.S.A.]. Reference materials from our laboratory were used for control of Ca, Mg and P analyses. The coefficients of variation for control materials of Zn, Fe, Ca, Mg and P were 3'1, 4'4,4,1, 3·7 and 5'4%, respectively. N analysis was performed by a micro-Kjeldahl technique (Technicon Auto Analyzer). In cereals, non-starch polysaccharide constituents (NSP) and lignin are the major components of 'dietary fibre'. These fibre fractions were measured according to method C of Theander and Westerlund16.

* Buttermilk manufactured from regular milk, heat treated at 90-91 °C for 3 min, inoculated with a mixed starter culture of Streptococcus lactis, Streptococcus cremoris, Streptococcus diacetylaclis and Leucollosloc cremol'is and cultured at 20-21 °C for 20-24 h. A typical product has a pH = 4'5. The fermented milk did not contain phytase activity.

192

B. KIVISTO ET AL.

Inositol tri-, tetra-, penta- and hexaphosphates of raw bran, extruded products and rice were determined according to Sandberg and Ahderinne 9 • The method included extraction of inositol phosphates with HCI, separation of the inositol phosphates from the crude extract by ionexchange chromatography and ion-pair C 18 reverse phase HPLC analysis using formic acid/methanol in the mobile phase. Phytate was also determined by an iron precipitation method according to Davies and Reid 16. Phytase activity in the extruded bran product was determined by analysis of phytate before and after incubation at optimal conditions for phytase. The samples were incubated at 55°C with ten volumes of water after adjustment of the initial pH to 4·5 with HCp7. After 30 min the pH had increased to 5·0 which is optimal conditions for wheat phytase 13 . After incubation for 17 h the product was freeze-dried before analysis of phytate. The effect of in vitro digestion on soluble Zn and added 65Zn was studied by a combination of the methods described by Hallberg and Bjom-Rasmussen 18 and Miller et al. 19 Weighed amounts of the raw bran or extruded products were transferred to six Erlenmeyer flasks. To two of the flasks 0·2 % (wIv) NaCI (50 ml) was added, to the other four flasks were added a simulated' gastric juice' mixture (50 ml) containing (per I) 0'32 g pepsin (Sigma No P·7012), sodium chloride (2 g) and 25 % (wIv) HCI (10 ml). To each flask was added 65Zn (0'02 MBq). Segments of dialysis tubing (Molecular weight cut off 6-8000, Spectrapor I, Spectrum Medical Industries, Los Angeles, U.S.A.) containing 15 ml of saline or 'gastric juice' without pepsin were placed in the two flasks with NaCI and in two of the flasks with 'gastric juice', respectively. The flasks were sealed with Parafilm and incubated in a 37°C shaking water bath for 30 min for the samples in saline and 2 h for the other samples. The dialysis tubes were removed and the Zn and 65Zn content determined. The content of the two remaining flasks was adjusted to pH 8 by adding drops of ammonium hydroxide (25 % wjv). Trypsin (30 mg; Sigma Chemicals No. T- 8253) was added and the flasks incubated at 37°C with dialysis tubing containing 15 ml of 'gastric juice' (without pepsin) adjusted to pH 8. The dialysis tubes were removed after 4 h and the content of Zn and 65Zn determined.

Statistical methods Student's I-test was used for statistical comparison.

Ethical considerations This project was approved by the Research Ethical Committee and the Isotope Committee at Sahlgren's Hospital.

Results The composition of the meals and extruded products are shown in Table II. The white bread contained no detectable phytate. Almost all Zn in the extruded products or their ingredients comes from the bran (less than 10 % from starch and gluten). Both the extruded bran products A, Band C as well as the rice contained inositol trio, tetra-, penta- and hexaphosphates. The results are shown in Table III. For comparison the amounts of phytate measured by iron precipitation according to Davies and Reid 16 are given. According to HPLC determinations extrusion cooking of product A reduced the inositol hexaphosphate content to 68 % of that in the raw bran but increased the amount of the penta-, tetra- and triphosphates to a corresponding degree so that the sum of inositol phosphates was constant. Also according to HPLC determinations, product C contained 11 % of the inositol hexaphosphate in product B. The absorption ofZn from the five test meals, estimated from retention measurements,

193

EXTRUSION COOKING AND ZINC ABSORPTION

TABLE I. The three extruded products used in the zinc absorption studies and the extrusion conditions Extruded product

Composition"

(%)

Added water (ml/kg)

Mass temperature (0C)

Mass pressure (MPa)

Screw speed (rev/min) - _ . _ - ~ _ . _ - ~ ~

A

B

C

II

11

30 60 10 20 70 10 20 70 10

bran starch gluten bran starch gluten bran b starch gluten

82

120

5·9

150

121

132

0·6

lSI

0

126

2·0

ISO

Dry weight basis. With reduced phytate content.

TABLE II. N, Ca, P, Mg, Zn, Fe, inositol hexaphosphate and dietary fibre contents of the meals

No. Meal composition

2 3 4 5

Fish+rice+ 27 g ingredients A Fish + rice + 27 g extruded prod A Milk + bread + 33 g extruded prod A Milk+ 150 g extruded prod B Milk+ 150 g extruded prod C

Ca P Mg Zn N Fe (g) (mmol) (mmo!) (mmol) (Ilffiol) (Ilmol) 3'2 0·5 3·1 0·2 1·4 0·2 1·0 2-8 1·0 2-8

1·5 0·3 1'5 0·3 5·8 0·4 5·0 1'2 5'7 1'2

10 5 10 4 6 5 6 15 6 IS

l-8 2'0 1'7 2·1 1·1 2·6

0·9 6'0 0·9 5'8

20 11 20 II 12 13 12 60 13 54

15 24 22 27 27 33 3 153 3 101

Inositol hexaphosphate (Ilmo1 )

..

Dietary fibre (g)

_--_.Jo6

77 437 77 300

1-6 3-6

366

0'7 4'4

3'4

1320

15

150

14

is shown in Table IV. There was no difference in Zn absorbtion from the two lunches with extruded product or non extruded ingredients respectively (meals nos. land 2). The amount of Zn absorbed from meal no. 4 was lower (P < 0·0 I) than that from the similar meal (no. 5) with reduced phytate content. There was no sign of phytase activity in either of the extruded products. Only small amounts of the total Zn (1-9%) and 65Zn (4-14 %) in the extruded products were dialysable in 0·2 % NaCl solution (Table V). After digestion at pH 2 in

194

B. KIVISTO ET AL.

TABLE III. Inositol phosphates" (!lmol/ g)b and phytate content (flmol/ g)h of bran products and rice

Ingredients A Extruded product Ingredients Band Extruded product Extruded product Rice

A C B

C

IP 3

IP4

IPs

IP.

Sum of IP 3 -IP.

Traces 0·8 Traces 0·2 1·0 0'5

Traces 2·1 Traces 0'3 0·3 0·4

1'7 4·9 1,2 1·8 0·2 0'6

16 II II 8'8 1'0 2'1

18 19 12 11 2'5 3·6

Phytate Davies and Reid method16 16 14 11 9·0 1·7 4·3

" IP. to lP a ", inositol containing three to six phosphates per inositol residue.

Dry weight basis.

b

TABLE IV. Zinc absorption from meals containing an extruded product or non-extruded products Zn absorption

!lmol

% Meal no.

n"

Mean (range)

S.E.M. b

8

36·6 (29'3-42,2)

2·0

2

8

31·8 (20'3-43'0)

3·1

3

9

6'5 (1'2-9'2)

0·8

4

8

6·2 (4'8-8'9)

0·6

5

8

17-6 (13'0-32'8)

2·4

Mean (range)

S.B.M. b

11·1 (8'9-12·8) 10,0 (6'3-13-4)

0·6

1-6

0·2

(0'3-2'2) 4·5 (3-5-6-4) 11·8 (8,7-21'9)

1·0

0·4 1·6

a n = number of subjects; the same eight subjects had meals nos. 1 and 2 while eight others had meals nos. 4 and 5. b

S.E.M. =

standard error of the mean.

•gastric juice' 55-88 % of the Zn and 65Zn were dialysable. After adjustment to pH 8 Zn and 65Zn again were practically non-dialysable. Discussion The absorption of zinc was in this study estimated from measurements of the whole body retention from a radio-zinc-Iabelled meal. With this technique most of the methodo-

EXTRUSION COOKING AND ZINC ABSORPTION

195

TABLE V. Dialysable Zn and B5Zn after in vitro digestion of raw bran and extruded products (as % of total Zn or 65Zn content). Each figure is the mean of duplicate determinations a After digestion in:

Raw bran Extruded product A Extruded product B Extruded product C

0·2% NaCI

' Gastric juice' pH2

'Gastric juice' pH8

Zn

65Z n

Zn

65Zn

Zn

B6Z n

0 7 1 9

9 4 14 14

80 79 88

63

56 58

0

I

78

6

5 0 8 2

• Standard error of the mean for duplicates

=

72

I

(}-4·4.

logical problems inherited in conventional techniques like the balance method are overcome. The intestine is the major route of endogenous excretion of zinc. Fecal zinc content is therefore a mixture of unabsorbed dietary zinc and excreted zinc that under conventional techniques can not be distinguished. The endogenous excretion of zinc amounts to 2-4 mg/day20 and is quantitatively important in relation to dietary zinc intake, which normally is in the range of 8-12 mg/day. However, compared to the total body, zinc pool it corresponds only to approximately 1 %. Thus variations in re-excretion of absorbed zinc isotope between or within individuals as well as normal variations in dietary zinc intake have minor effects for the evaluation of retention measurements. Therefore, instead of increasing the radiation dose by determination of rate of excretion after intravenous administration to each individual the average excretion rate in a group of subjects of similar age was used l l • The rate of zinc excretion forms a biexponential function. It corresponds to a mean excretion of 13·4 % during the first 14 days and an additional 6·4 % is excreted during the following 14 daysll. Thus, the allowance for re-excretion of absorbed isotope is relatively small. This mean retention function has been verified by other research groups5. The use of an extrinsic label to study absorption assumes that isotopic equilibration takes place between the label and the endogenous zinc in the meal. The in vitro digestion study showed an almost identical behaviour in 'gastric juice' of endogenous Zn and added 65Zn and indicates a similar degree of isotopic exchange in the non-processed and extruded products. In saline, the solubility of both endogenous Zn and added 65Zn was low, making the agreement seem less. The observations including the high solubility at low pH, indicate that the extrinsic labelling technique is a valid method for Zn absorption. Furthermore, an incomplete exchange, where only a part of the endogenous Zn was labelled, would overestimate rather than underestimate the degree of absorption as the fractional absorption of Zn depends upon the total content of Zn21.22. The low absorption of Zn observed from meals nos. 3 and 4 suggests the presence of some factor depressing Zn absorption. Phytic acid has been shown to have such an etfect12 . We have previously studied a meal with a similar amount (10 g) of uncooked wheat bran as in meal no. 3 in this study12. The Zn absorption observed in that study

196

B. KIVISTO ET AL.

was 9·6% (S.E. = 0'5) which is significantly different (P < 0'01) from that from meal no. 3 in the present study. These results indicate that the low Zn absorption from meal no. 3 is not explained by the phytic acid content alone but that extrusion cooking caused a decrease in Zn absorption. A possible explanation for this is the absence of phytase activity in the extruded product. Previous studies have indicated that phytate of raw bran containing phytase activity can be hydrolysed in the stomach and small intestine 2 :J. In a previous study on extruded product A, using a balance technique and ileostomized subjects, we found a lower digestibility of phytate in the extruded product compared to the ingredients 7. 24 and also an adverse effect of extrusion cooking on 'apparent' absorption ofZn and Mg 7 • In the present study, when the phytic acid content was reduced before the extrusion cooking (meal no. 5), Zn absorption was greatly improved. When the extruded product or its ingredients was served with a cooked meal there was no difference in Zn absorption. This can probably be explained by the high protein content of the meal, as it has been shown earlier that a high protein content improves the absorption of Zn from a phytic acid-containing meal 21 • A possible explanation for this observation is that protein digestion products, e.g. peptides and amino acids, facilitate Zn absorption and could also compete with complexing substances for Zn binding. Another important difference between the two types of meals is the Ca content; in animal studies Ca has been shown to reduce Zn absorption from phytic acid-containing meals or diets 25 • The phytic acid: Zn molar ratio has been suggested as a predictor of Zn bioavailability 26. In a study on rats Morris and Ellis 27 found that a phytate: Zn molar ratio exceeding 15 depressed growth. However, in the present study the phytate: Zn molar ratio was 12-18 for meals nos. 1--4 and only 2 for meal no. 5 but still Zn absorption was much greater from meals nos. 1 and 2 than from meal no. 5 so that for these meals the phytic acid: Zn molar ratio cannot be used to predict Zn availability. In rats the dietary Ca level has been found greatly to influence the availability of Zn when phytate is present 2E • [Ca][phytate]/[Zn] ratio has been shown to relate to the weight gain of rats with a value above 3·5 making the available zinc supply inadequate to maintain growth 29 • However, in the present study the [Ca][phytate]/[Zn] ratio for meals nos. 1-5 was 1'0,0,7,3'7,1'5 and 0'2, respectively corresponding to 36,6,31,8,6'5,6·2 and 17·6% absorption making also this ratio unsuitable for predicting 2n availability. The values for phytate obtained with the iron precipitation method 16 approximately equals the amount ofphytate determined by HPLC in the bran. However, in processed food containing considerable amounts of inositol phosphates with lower degrees of phosphorylation than the hexaphosphate, the phytate content may be overestimated using precipitation methods as the penta-, tetra- and triphosphates are partly precipitated 30. The effect of penta-, tetra- and lower phosphates of inositol on the bioavailability of minerals in man is poorly known. Uinnerdal et al. 10 found an inhibitory effect on Zn and Ca uptake in rats for hexa- and penta-phosphates of inositol, but not for tetra- and triphosphates of inositol. This needs to be further investigated in man. Their observations as well as the results from this study also emphasizes the need for appropriate analyses of phytic acid in processed food, when used in mineral absorption studies. We conclude that extrusion cooking of a high fibre cereal product has a slight negative effect on Zn absorption and attribute it to the absence of phytase activity in the extruded

EXTRUSION COOKING AND ZINC ABSORPTION

197

product. rt is however possible that the use of other extrusion conditions may have other effects on trace element availability. Since there is an increasing use of extrusion cooking for processing basic foods, e.g. weaning foods in developing countries, gruels, breakfast cereals and bread, a thorough knowledge of its effect on the nutritional value is essential. The authors wish to thank Ms Annette Almgren for excellent technical assistance. We also thank Dr Lena Jonsson and Dr Yngve Andersson at the Swedish Food Institute (SIK) for extruding the . bread '. Research was supported by grants from the National Swedish Board for Technical Development (project nos. 79-5225 and 79-5226).

References I. 2. 3. 4.

5. 6. 7. 8. 9. 10. I I. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25.

26. 27. 28. 29. 30.

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