Hydrothermal Processing of Barley (cv. Blenheim): Optimisation of Phytate Degradation and Increase of FreeMyo-inositol

Journal of Cereal Science 29 (1999) 261–272 Article No. jcrs.1998.0239, available online at http://www.idealibrary.com on

Hydrothermal Processing of Barley (cv. Blenheim): Optimisation of Phytate Degradation and Increase of Free Myo-inositol E.-L. Bergman∗, K. Fredlund∗, P. Reinikainen† and A.-S. Sandberg∗ ∗ Chalmers University of Technology, Department of Food Science, c/o SIK, Box 5401, S-402 29 Go¨teborg, Sweden; † Oy Lahden Polttimo AB, P.O. Box 22, Fin-151 41 Lahti, Finland Received 19 January 1998

ABSTRACT Optimal conditions were developed for hydrothermal processing of whole barley kernels (cv. Blenheim) to degrade phytate (myo-inositol hexaphosphate) and to increase the content of free myo-inositol. The hydrothermal treatment comprised of two wet steeps, where lactic acid solution of different concentrations was used, and two dry steeps followed by successive drying. Experiments were performed as a central composite design and evaluated by multiple linear regression. The variables in the experiments were temperature in the first wet and dry steep (T1), temperature in the second wet and dry steep (T2) and lactic acid solution concentration in both wet steeps (C) and mathematical models were developed in these variables. Optimal conditions for maximal phytate degradation and for maximal increase of free myo-inositol were T1=48 °C, T2=48–50 °C and C=0·8%, at these conditions the amount of phytate was reduced by 95–96% and the free myo-inositol concentration was increased from 0·56 to 2·45 lmol/g d.m. We conclude that this hydrothermal process can be used to produce a barley product (cv. Blenheim) with a low phytate content and a high level of free myo-inositol.  1999 Academic Press Keywords: barley, phytate, myo-inositol, hydrothermal processing, mathematical modelling.

INTRODUCTION An adequate mineral absorption is important especially for infants1, children, elderly people and people in clinical situations. Wholemeal cereal products have a high mineral content, but they also contain considerable amounts of phytic acid (myoinositol hexaphosphoric acid) which chelates with minerals such as calcium2, iron3–5 and zinc3,6,7,  : cv.=cultivar; d.m.=dry matter; IP6–IP3=inositol hexa-, penta-, tetra- and triphosphate; MI=free myo-inositol; n.d.=not detectable. ∗Corresponding author: E.-L. Bergman. Tel: +46 31 355626; Fax: +46 31 833782; E-mail: eva-lotta. [email protected] 0733–5210/99/030261+12 $30.00/0

forming insoluble complexes (phytates) rendering minerals unavailable for absorption in the human intestine. One way of solving this during the food process is to activate the intrinsic enzyme phytase which degrades phytate to free myo-inositol and inorganic phosphate via intermediate myo-inositol phosphates (penta- to monophosphates)8. In some cases, this can also occur in the alimentary tract if there is an active dietary phytase9,10. When phosphate groups are removed from phytate the mineral bioavailability is increased7,11,12. By malting, milling and soaking or just soaking of flours at optimal conditions for endogenous phytase (pH 5, 55 °C)13, phytate in wheat, barley and rye can be completely hydrolysed14,15. Studies in animals indicate that myo-inositol is  1999 Academic Press

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an essential nutrient16,17. Too low amounts in the diet cause deficiencies such as accumulation of triacylglycerols and abnormal fatty acid metabolism. If the deficiency is prolonged, the animals progressively decrease in weight and suffer from alopecia (loss of hair), skin lesions, exhaustion, and this ultimately leads to death. However, in humans myo-inositol is considered to be only a semiessential nutrient18; although the high content of free myo-inositol in human milk (331±81 mg/L) suggests that it is an important nutrient for infants19. The concentration of free myo-inositol in human milk is several times higher than in various infant formulae and parenteral nutrition solutions19. In Sweden, products for infants are based on flour from cereals but they can also contain legumes. Myo-inositol in cereals and legumes is mostly present as myo-inositol hexaphosphate and is unavailable for absorption by humans. More studies need to be done to evaluate the nutritional importance of myo-inositol and if it should be recommended to be included in infant formulae. Even now myo-inositol is added at a concentration of 0·01% to some infant formulae20 to prevent a possible deficiency. Before the modern milling industry prevailed, hydrothermal processes were widely used prior to the dehulling and milling of cereal seeds21,22. The grains were, depending on variety, first moistened or left to soak for several hours, then dried before being pounded23. The husk was then removed with a domestic pestle. The process of moistening, drying, pounding and separating had to be repeated several times. Treatment of seeds in this way was believed to improve the dehulling and storing properties of the seed since it removed the husk without damaging the protein layer. Our previous studies24 show that phytate is also degraded during hydrothermal processing of wheat, rye, barley and oats. In the present study barley was used as there is a long tradition of its use in the Swedish household and human studies have shown that barley has health effects such as reduction of postprandial glucose, insulin and serum lipid levels25, although the consumption of barley in Sweden today is low; about 0·4 kg per capita. Barley is a tolerant crop and can be adapted to different climates. The countries of the European Union (EU) produced 39 tonnes of barley during 1994 and Germany, France, Spain and the United Kingdom were the largest producers25. It is also a crop which should adapt well to the hydrothermal

process since this process resembles malting where barley is the most often used cereal. The aim of this study was to optimise a hydrothermal process to achieve a barley product (cv. Blenheim) with a low level of phytate and a large amount of free myo-inositol and hence a high bioavailability of minerals and myo-inositol. It was also intended to produce a hydrothermal product with high phytase activity. EXPERIMENTAL Barley seeds Whole barley kernels (cv. Blenheim) of 1994 crops were provided by Skanska Lantmannen (Sweden) and used throughout the study. The barley contained 12·8% protein, 4·0% fat, 12·8% water, 69·3% carbohydrates by difference, 11·5 mg/ 100 g sodium, 493 mg/100 g potassium, 65·4 mg/ 100 g calcium, 5·8 mg/100 g iron, 126 mg/100 g magnesium, 0·5 mg/100 g copper, 2·8 mg/100 g zinc, 1·0 mg/100 g manganese and 356 mg/100 g phosphorus. The kernels were not heat treated before being stored at room temperature until processed. The hydrothermal process The process (see flow chart in Fig. 1) was executed in a laboratory plant at Oy Lahden Polttimo AB, Finland. The whole kernels were washed in cold water and soaked in 1·5 volumes (Experiments 1–26) or 3·2 volumes (Experiments 29–31) of lactic acid solution (0–0·8%, Tables I and II) for 1 h at 45–70 °C (Tables I and II). Superfluous lactic acid solution was drained and the seeds were dry steeped for 5 h at 45–70 °C (Tables I and II). The seeds were then steeped in 1·5 volumes (Experiments 1–26) or 3·2 volumes (Experiments 29– 31) of lactic acid solution (0·0–0·8% v/w, Tables I and II) for a further hour at 40–75 °C (Tables I and II), superfluous lactic acid solution was drained and dry steeping for 15 h followed at 40–75 °C (Tables I and II). The processed barley was airdried at 50 °C for 8 h, followed by a progressive increase of temperature to 80 °C during 2 h and drying was continued at 80 °C for 6 h. Experiments 27 and 28 were processed as indicated in Table II. Dried end products (DP) from all experiments were analysed. Samples from Experiments 1–24 (Fig. 1) were collected, cooled and freeze-dried after the first wet steep (A I), before the second

Hydrothermal processing of barley (cv. Blenheim)

263

Wet steep I

the pH to an optimal pH for phytate degradation and increase of free myo-inositol during the hydrothermal process. Lactic acid is an acid with a neutral flavour and is accepted as a food ingredient. Our previous studies24 showed that incubation of naked barley (cv. Taiga) in lactic acid or acetate resulted in a more extensive phytate degradation than incubation in water, citrate or citric acid.

Lactic acid, 0–0.8% v/w T1 = 45–70°C (1 h)

Experimental design

Raw material

Cold water wash

AI Dry steep I T1 = 45–70°C (5 h) B II

Wet steep II Lactic acid, 0–0.8% v/w T2 = 40–75°C (1 h) A II

Dry steep II T2 = 40–75°C (15 h) BD Drying 50°C for 8 h 50–80°C for 2 h 80°C for 6 h (16 h)

Product

The process was optimised with respect to three variables (Table I): temperature at the first wet and dry steep (T1, °C), temperature at the second wet and dry steep (T2, °C) and lactic acid solution concentration used in the wet steep steps (C, % v/ w). Five screening experiments (Experiments 1–5, Table II) were carried out to ensure that the factors were sufficiently varied to give significantly different results. The results were satisfying and 18 more experiments (Experiments 6–23, Table II) were carried out as a central composite design with four replicates at the centre point. A verification experiment was carried out at predicted optimal conditions in the experimental domain (Experiment 24, Table II). Experiments 25–31 were carried out outside the experimental domain for reasons explained in the Results section. Real and scaled values of the variables are given in Table I. The experimental conditions at the centre point of the central composite design were T1= 55 °C, T2=65 °C and C=0·4%. The scaled values for the variables in the design were x1=(T1−55)/ 5, where T1 was varied between 45 and 65 °C; x2=(T2−65)/5 where T2 was varied between 55 and 75 °C; x3=(C−0·4)/0·2 where C was varied between 0 and 0·8% (v/w).

DP

Determination of inositol phosphates Figure 1 Flow chart of the hydrothermal process. Samples were taken out after the first wet steep (A I), before the second wet steep (B II), after the second wet steep (A II), before drying (BD) and as dried end product (DP).

wet steep (B II), after the second wet steep (A II) and before drying (BD). Freeze-dried samples and dry products were stored at a temperature below −18 °C until analysed for inositol phosphates, free myo-inositol or release of inorganic phosphorus as an indirect measurement of phytase activity. Lactic acid was used in the wet steeps to lower

Before analyses the samples were thawed and then ground in a grinder (Braun AG type: 4041) for 30 s. Approximately 0·4 g of the ground samples were analysed in duplicate for dry matter (d.m.) using a moisture balance (Precisa HA 300, Zu¨rich, Switzerland). Duplicate samples (0·5 g d.m.) of ground seeds were extracted with 0·5  HCl (20 mL) for 3 h. The extracts were centrifuged and the supernatants were decanted, frozen overnight, thawed and centrifuged again. After the second centrifugation, 15 mL of the supernatant was evaporated to dryness by a flow of air and then

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Table I

Scaled and real values of independent variables used for optimising a hydrothermal process with respect to degradation of phytate and increase of free myo-inositol Real value

Independent variables

Symbols

Scaled value

Levels

Symbols

Levels

Temperature at wet and dry steep I (°C)

T1

65 60 55 50 45

x1

2 1 0 −1 −2

Temperature at wet and dry steep II (°C)

T2

75 70 65 60 55

x2

2 1 0 −1 −2

Lactic acid concentration in wet steep I and II (% v/w)

C

x3

2 1 0 −1 −2

redissolved in 0·025  HCl (15 mL). The inositol phosphates were separated from the crude extract by ion exchange chromatography according to Sandberg and Ahderinne26. For steep water samples, 20 mL were put directly on columns with ion exchange resin. Inositol hexa-, penta-, tetraand triphosphates were determined by ion-pair C 18 reverse-phase HPLC using formic acid/ methanol and tetrabutylammoniumhydroxide in the mobile phase12,26. The HPLC comprised of an HPLC pump (Waters Model 510, Waters Associates Inc., Massachusetts), a C 18 Chromasil (5 l) column 2 mm i.d. and a refractive index detector (ERC-7510 RI-detector Erma Optical Works Ltd., Japan). The flow rate was 0·4 mL/ min. Retention times and peak areas were measured by the laboratory data system, HP 3350 (Hewlett Packard Ltd.). Injections were made with a 20 lL loop. 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±.. of wheat bran samples was 46·9±1·5 lmol/g d.m. myo-inositol hexaphosphate. 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±.. were 10·3±0·2 and 9·9±0·35 lmol/g d.m. myo-inositol hexaphosphate. The accuracy of the HPLC method was estimated from these results to be about ±3%. To ensure that the method did not change over

0·8 0·6 0·4 0·2 0·0

time a duplicate sample of oat flakes, ground, extracted and analysed as described above, was analysed together with each round of analyses.

Determination of free myo-inositol Before analyses the samples were thawed and then ground in a grinder (Braun AG type: 4041) for 30 s. Approximately 0·4 g of the ground samples were analysed in duplicate for dry matter (d.m.) with a moisture balance (Precisa HA 300, Zu¨rich, Switzerland). Then 0·1 mg of internal standard (pentaerythritol) was added to triplicate samples of ground seeds (0·5 g d.m.). Ten millilitres of deionised water was added and the samples were heated to 92 °C for 30 min. The samples were magnetically stirred for 10 min, ultrasonically treated for 20 min, stirred for another 10 min and centrifuged. The supernatants were frozen for at least 1 h, thawed and centrifuged. An aliquot (1 mL) of the supernatants were evaporated to dryness and then silylated according to SantaMaria et al.27. The silylated samples were analysed for free myo-inositol by high-temperature capillary gas chromatography. A 0·01 mg internal standard (pentaerythritol) was added to 2 mL of the steep water samples. The samples were evaporated to dryness, and treated and analysed as above. Analyses were performed on a Carlo Erba HRGC-5300 HT Mega Series (Milan, Italy) gas

Hydrothermal processing of barley (cv. Blenheim)

Table II

Process conditions, content of inositol phosphates and free myo-inositol in end productsa Variables

Experiment Raw material 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27c 28d 29e 30e 31e

265

Responses

T1 (°C)

T2 (°C)

C (%)

IP3b

IP4b

IP5b

IP6b

% degraded IP6b

MIb

— 55 55 55 70 70 55 60 45 50 50 55 65 55 60 60 55 55 50 60 55 55 50 55 45 48 48 48 48 48 48 48

— 55 55 55 70 70 65 60 65 70 60 75 65 65 60 70 65 65 70 70 55 65 60 65 55 50 48 — — 40 50 48

— 0·0 0·1 0·6 0·1 0·6 0·4 0·6 0·4 0·2 0·6 0·4 0·4 0·4 0·2 0·2 0·0 0·4 0·6 0·6 0·4 0·8 0·2 0·4 0·8 0·8 0·8 0·8 0·8 0·8 0·8 0·8

0·36 1·44 1·66 2·92 0·66 0·55 1·11 1·10 1·14 0·64 2·28 0·95 0·79 1·08 1·06 0·98 0·94 1·28 1·07 1·28 1·95 1·64 1·00 1·24 2·37 1·73 1·84 1·07 0·94 0·49 1·38 1·89

0·12 0·97 1·30 2·07 0·71 0·98 1·46 1·73 1·58 1·05 2·05 0·88 1·29 1·28 1·50 1·14 0·84 1·43 1·15 1·37 1·70 1·84 1·25 1·41 1·74 0·79 1·02 0·83 1·51 0·38 0·67 0·87

0·44 0·62 0·68 0·47 1·71 1·60 0·80 1·04 0·97 1·08 0·52 1·08 1·11 0·81 0·92 1·07 0·81 0·84 1·08 0·96 0·44 0·77 1·00 0·76 0·41 0·15 0·27 0·54 1·36 1·00 0·14 0·18

13·88 5·51 4·84 1·86 8·75 8·20 4·94 5·12 6·05 6·97 2·53 4·72 6·87 4·91 5·01 5·70 5·72 4·40 5·74 4·96 2·28 3·22 6·18 4·59 1·70 0·66 1·29 3·80 6·60 7·31 0·50 0·84

— 60 65 87 37 41 64 63 56 50 82 66 51 65 64 59 59 67 59 64 84 77 55 68 88 95 91 73 52 47 96 94

0·56 0·84 1·16 0·94 0·31 0·31 0·98 0·88 0·62 0·69 0·98 1·03 0·53 0·98 0·81 0·67 0·94 1·10 0·80 0·76 1·32 1·11 0·88 1·13 1·53 2·23 2·45 2·68 1·03 1·88 2·00 2·11

Values are lmol/g dry matter. IP6–IP3: inositol hexa-, penta-, tetra- and triphosphate; MI: free myo-inositol. c The second wet steep excluded and the first dry steep prolonged to 21 h. d The second wet steep excluded and the first wet steep prolonged to 5 h and the dry steep prolonged to 17 h. e 3·2 volumes of lactic acid were used in the process. a

b

chromatograph fitted with an on-column injector and a FID detector (temperature 400 °C). An HT polyimide-fused silica column (25 m×0·32 i.d., Quartz and Silice, France) was static-coated with cross-linked SE-54 (Alltech Associates, Deerfield, IL, U.S.A.) according to the method of Blomberg et al.28; the film thickness was 0·05 lm. Helium was used as the carrier gas at a linear gas velocity of 60 cm/s at 60 °C. A high-capacity gas purifier (Oxisorb, Messer Griesheim, Germany) and a 5˚ molecular sieve (Alltech Associates) were used A in the carrier gas line. Samples were injected oncolumn at 60 °C. A linear temperature program (20 °C/min) was run up to 389 °C. Integration

was performed with a PC-pack Softran Kontron Instrument (Milan, Italy). Release of inorganic phosphorus Release of inorganic phosphorus during incubation at optimal conditions for phytase activity (pH 5, 55 °C)13 was used as an indirect measurement of phytase activity and was measured according to Fiske and Subbarow29 with some modifications. Phytase activity was calculated as released inorganic phosphorus per minute and gram d.m. Before analyses the samples were thawed and then ground in a grinder (Braun AG

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266

type: 4041) for 30 s. Approximately 0·4 g of the ground samples were analysed in duplicate for dry matter (d.m.) with a moisture balance (Precisa HA 300, Zu¨rich, Switzerland). To duplicate samples of ground seeds (0·4 g d.m.), 3% trichloraceticacid (8 mL) and citrate buffer (20 m, 4 mL) pH 5 were added. After 5 min, de-ionised water (12 mL) was added. The same samples were soaked in citrate buffer (20 m, 4 mL) pH 5 at 55 °C for 1 h. Three percent trichloraceticacid (8 mL) was added to stop enzymic activity and de-ionised water (12 mL) was added after 5 min. The samples were centrifuged and 0·5–1·5 mL of the supernatants were mixed with 4·0 mL of 2·5% sulphuric acid, 1 mL of a 50:50 mixture of ammoniummolybdat and dimethylsulphoxide and 0·25 mL of aminonaphtolsulphonic acid. Inorganic phosphorus was determined spectrophotometrically (Hitachi, model 101). For steep water samples, an aliquot (10 mL) was added to 1·4 mL 10 lmol/mL myoinositol hexaphosphoric acid, the pH was adjusted to 5·0 and the samples were incubated and analysed as above. Data analysis Mathematical models were fitted and results were statistically evaluated by multiple linear regression30. The observed values of myo-inositol hexaphosphate and free myo-inositol [yobs, equation (1)] in the dried products were fitted to mathematical models in three independent processing variables expressed in scaled values: yobs=b0+b1x1+b2x2+b3x3+ b11x1x1+b22x2x2+b33x3x3+ b12x1x2+b13x1x3+b23x2x3+e

(1)

e=yobs−ycalc

(2)

In these equations b0 is a constant; b1, b2 and b3 express the main effect of each process variable; b11, b22 and b33 are the square coefficients; b12, b13 and b23 show the interactions between the variables. The difference between the measured values (yobs) and the calculated values (ycalc) gives the residual in equation (1)31. Both the real values and the scaled values of the experimental variables are shown in Table I. As the experiments in this study were carried out as a central composite design (see Experimental design) it was possible to estimate the coefficients in equation (1) by multiple linear regression30. This is due to the fact that the

variable settings are varied independently between the experiments in a specific manner. The coefficients in equation (1) are estimated with multiple linear regression to minimise the sum of squares of the residual (SSe), equation (3): n

SSe=

n

e =(y 2

1

obs

−ycalc)2

(3)

1

where n=the number of experiments. SSe is the sum of two types of noise; SSlof, which is due to the incapability of the model to exactly approximate the response, and SSperr which is due to the pure experimental error which is always present in experimental work. The pure error can be estimated by repeating one experiment several times and/or repeating some or all experiments. In this study we chose to repeat the experiment in the centre point of the design four times (Experiments 6, 13, 17 and 23). The mathematical models were used to create response surface plots to see how the content of phytate (myo-inositol hexaphosphate) and free myo-inositol varied with the experimental variables and to predict optimal conditions for degradation of myo-inositol hexaphosphate and increase of free myo-inositol. The explained variance (R2) and the predictive capacity (Q2) were calculated for the models to estimate the reliability of the models. R2 is the variation of the response explained by the model and Q2 is the fraction of the variation of the response that can be predicted by the model. Q2 must exceed 0·5 if any conclusions are to be drawn from the model and the model is generally considered excellent if both R2 and Q2 exceed 0·931. RESULTS Phytate A mathematical model was fitted to phytate concentrations in the hydrothermal barley products from Experiments 1–26 (Table II) by using multiple linear regression30. Since the square terms T2T2 (x2x2) and CC (x3x3) and the interaction term T2C (x2x3) did not influence the model significantly (P>0·05) and the model improved when these terms were removed they were excluded from the model. The achieved model, expressed in scaled variables, was: Y(lmol/g d.m. myo-inositol hexaphosphate)=

Hydrothermal processing of barley (cv. Blenheim)

Table III

267

Analysis of variancea (ANOVA) for mathematical models based on the results of phytic acid and myo-inositol Myo-inositol

Phytic acid

Total Constant Total corrected Regression Residual Lack of fit (Model error) Pure error (Replicate error)

DFb

SSc

DF

SS

26 1 25 6 19 16 3

683·868 579·238 104·63 96·234d 8·395 8·192 0·203

25 1 24 7 17 14 3

30·86 25·08 5·78 5·540d 0·239 0·221 0·019

a

Calculated using the computer program MODDE 3.0 (Umetri AB, Umea˚, Sweden). Degrees of freedom. c Sum of squares. d Significant at the 0·001% level (P<0·00001). b

4·56−0·01x1+0·50x2−0·76x3+ 0·47x1x1−0·24x1x2+0·24x1x3 75

(a) 5.4

5.4 4.9

4.9

3.7

4.3

4.3

T2 (°C)

70 3.2 65 2.6 60 2.1 55 45

75

50

55 T1 (°C)

65

60

(b) 0.8

0.4 0.6 0.8

70 T2 (°C)

The temperature during the second wet and dry steep (T2, x2), the lactic acid concentration during the wet steep steps (C, x3), the square term of the temperature during the first wet and dry steep (T1T1, x1x1) and the interaction term between the temperature used in the first wet and dry steep and the lactic acid concentration during the wet steep steps (T1C, x1x3) significantly influenced the content of phytate (myo-inositol hexaphosphate) in the end product (P<0·04). The model showed no significant lack of fit (Table III), the explained variance (R2) was 0·92 and the predictive capacity (Q2) was 0·82, thus conclusions can be drawn from the model. The response surface plot based on the model [Fig. 2(a)] showed that optimal conditions for phytate degradation probably were outside the experimental domain regarding T2. Therefore T2 should be set below 55 °C and this was done in Experiments 25, 26 and 29–31. The most extensive phytate reduction was achieved at T1=48 °C, T2= 50 °C and C=0·8%. The phytate content was reduced by 95–96% under these conditions (Experiments 25 and 30, Table II) and they are thus considered optimal for phytate reduction in barley (cv. Blenheim). Experiments 26, 27 and 28 were carried out under the same conditions (Table II). However, the second wet steep was excluded in Experiments 27 and 28, and the dry steep was prolonged to 21 h in Experiment 27 and the wet steep and the dry steep were prolonged to 5 h and 17 h, respectively, in Experiment 28. The results from these three experiments are shown in Table II.

1.0

65

1.2 60 1.4 55 45

1.6 50

55 T1 (°C)

60

65

Figure 2 Response surface plot at 0·8% (v/w) lactic acid concentration for (a) phytate and (b) free myo-inositol. Values are lmol/g d.m. phytate and free myo-inositol.

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Table IV

Process conditions and content of free myo-inositola Free myo-inositol

Variables Experiment 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27c 28d

T1 (°C)

T2 (°C)

C (%)

A Ib

B IIb

A IIb

BDb

DPb

55 55 55 70 70 55 60 45 50 50 55 65 55 60 60 55 55 50 60 55 55 50 55 45 48 48 48 48

55 55 55 70 70 65 60 65 70 60 75 65 65 60 70 65 65 70 70 55 65 60 65 55 50 48 — —

0·0 0·1 0·6 0·1 0·6 0·4 0·6 0·4 0·2 0·6 0·4 0·4 0·4 0·2 0·2 0·0 0·4 0·6 0·6 0·4 0·8 0·2 0·4 0·8 0·8 0·8 0·8 0·8

0·29 0·31 0·32 0·32 0·31 0·50 0·70 0·48 0·49 0·55 0·53 0·46 0·64 0·61 0·63 0·61 0·65 0·57 0·70 0·59 0·55 0·52 0·66 0·59 — 0·60 0·60 0·65

0·87 0·83 0·89 0·32 0·37 1·07 0·95 0·57 0·72 0·85 1·15 0·59 1·20 0·96 0·73 1·17 1·27 1·18 1·02 1·14 1·21 0·72 1·28 1·05 — 1·19 1·19 —

0·88 0·78 1·01 0·31 0·33 0·93 0·74 0·48 0·39 0·76 — 0·47 0·91 0·81 0·66 1·09 1·03 0·73 0·81 1·01 1·02 0·70 1·06 0·95 — 1·34 — —

1·14 1·05 1·24 0·29 0·30 — 0·76 — 0·65 1·03 — 0·51 1·00 1·02 0·74 1·20 1·08 0·80 0·78 1·25 1·09 0·96 1·12 1·43 — 2·43 2·32 1·04

0·84 1·16 0·94 0·31 0·31 0·98 0·88 0·62 0·69 0·98 1·03 0·53 0·98 0·81 0·67 0·94 1·10 0·80 0·76 1·32 1·11 0·88 1·13 1·53 2·23 2·45 2·68 1·03

Values are lmol/g dry matter. After the first wet steep (A I), before the second wet steep (B II), after the second wet steep (A II), before drying (BD) and dried end product (DP). c The second wet steep excluded and the first dry steep prolonged to 21 h. d The second wet steep excluded and the first wet steep prolonged to 5 h and the dry steep prolonged to 17 h. a

b

Steep waters from four experiments (Experiments 24, 26, 28 and 29, Table IV) were analysed for phytate. The phytate content in all samples was found to be low. Free myo-inositol A mathematical model was fitted to free myoinositol in the hydrothermal barley products from Experiments 1–26 (Table II) by using multiple linear regression30. Experiment 3 was found to be an outlier and was therefore excluded from the calculations. Since the square term CC (x3x3) and the interaction term T1C (x1x3) did not influence the model significantly (P>0·05) and the model improved when these terms were removed they

were excluded from the model. The achieved model, expressed in scaled variables, was: Y(lmol/g d.m. free myo-inositol)= 0·94−0·01x1−0·08x2+0·05x3− 0·09x1x1−0·05x2x2+0·07x1x2−0·06x2x3 The temperature during the second wet and dry steep (T2, x2), the lactic acid concentration during the wet steep steps (C, x3), the square term of the temperature during the first wet and dry steep (T1T1, x1x1), the square term of the temperature during the second wet and dry steep (T2T2, x2x2), and the interaction term between the temperature used in the second wet and dry steep and the lactic acid concentration during the wet

Hydrothermal processing of barley (cv. Blenheim)

steep steps (T2C, x2x3) significantly influenced the content of free myo-inositol in the end products (P<0·05). The model showed no significant lack of fit (Table III), the explained variance (R2) was 0·96 and the predictive capacity (Q2) was 0·92, thus the model is excellent and conclusions can be drawn from it. The response surface plot [Fig. 2(b)] indicated that optimal conditions were outside the experimental domain regarding T2. Therefore T2 should be set below 55 °C. In Experiments 25, 26 and 29–31, T2 was set below 55 °C. Maximal concentration of free myo-inositol in the end product, 2·45 lmol/g d.m., was achieved at T1=48 °C, T2=48 °C and C=0·8% (Experiment 26). Experiments 26, 27 and 28 were carried out under the same conditions (Table II). However, the second wet steep was excluded in Experiments 27 and 28, and the dry steep was prolonged to 21 h in Experiment 27 and the wet steep and the dry steep were prolonged to 5 h and 17 h, respectively, in Experiment 28. The results from these three experiments are shown in Table II. The end product from Experiment 27 contained a higher level of free myo-inositol than the end products from Experiments 26 and 28. Table IV shows how the content of free myoinositol changed during the hydrothermal process in Experiments 1–23. The free myo-inositol levels in Experiments 1–23 did not change significantly (P>0·05) during the first wet steep. During the first dry steep the levels increased significantly (P<0·05) in all experiments except in Experiments 4, 5, 7 and 15. During the second wet steep, all levels of free myo-inositol decreased except in Experiments 1 and 3. The second dry steep increased levels significantly (P<0·05) in 11 out of 26 samples. Drying did not influence the level of free myo-inositol in most cases. Analyses of the steep waters from Experiments 24, 26, 28 and 29 showed small amounts of free myo-inositol in the steep waters (Table V).

Release of inorganic phosphorus Samples from five experiments (7, 12, 17, 20 and 23, Table VI), steep water from Experiments 24, 26 and 28 (Table V) and the raw material were analysed for phytase activity measured indirectly as released inorganic phosphorus during incubation at optimal conditions for phytase activity. Analyses showed that the phytase activity of the barley decreased gradually in all experiments dur-

269

ing the process. The phytase activities in the end products were 4–13% of the phytase activity in the raw material. The phytase activity in steep water from the first wet steep in Experiment 24, was approximately 5% of the phytase activity in the raw material and approximately 8–9% of the phytase activity in Experiments 7, 17, 20 and 23 after the first wet steep. Phytase activities in the second steep waters from Experiments 24 and 26 were insignificant.

DISCUSSION AND CONCLUSIONS The phytate contents in the products achieved at optimal conditions for maximal phytate reduction in the hydrothermal process (Experiments 25 and 30, T1=48 °C, T2=50 °C and C=0·8%) were low enough (0·50–0·66 lmol/g d.m.) not to be considered to significantly affect zinc absorption3, and low enough not to have a strong negative effect on iron absorption3,4. It is clear from the changes in phytate content during the process that the greatest and fastest phytate reduction took place during the first wet steep (25–36% reduction, Fig. 3). In samples processed at 55 °C, 16–29% of the phytate was reduced during the first dry steep, about 9–10% during the second wet steep, and 15–22% during the second dry steep. Barley phytase is probably deactivated at a temperature >65 °C, depending on the moisture content of the seeds (Table VI, Fig. 3). According to Lee32 phytase extract from malted barley was completely inactivated after 15 min at 60 °C. Optimal conditions for increase of free myoinositol were T1=48 °C, T2=48 °C and C=0·8% (Experiment 26). The level of free myo-inositol was 4·4 times higher in the product from Experiment 26 (2·45 lmol/g d.m., i.e., 0·44 mg/g d.m.) than in the raw material. If this product is used in foods for infants and children about 12 g of hydrothermally treated barley and 8 g milkpowder are used per portion of ready-to-eat formula. This will give 7·0 mg (12×0·44+8×0·2233) free myoinositol/portion. For comparison, infant formulae for 0–6 months old children contain 6·6 mg free myo-inositol/portion according to a baby food manufacturer (Semper AB, Sweden). Comparing Experiment 26 with Experiment 27 (see The hydrothermal process for details), where the second wet steep was excluded, showed that there was a higher amount of phytate and free

E.-L. Bergman et al.

270

Table V Experiment

Inositol phosphatesa, free myo-inositola and phytase activityb in steep water

Steep water

IP3c

IP4c

IP5c

IP6c

Myo-inositol

Phytase activity

I II I II I I II

0·162 0·135 0·036 0·055 0·010 0·005 0·003

0·048 0·045 0·004 0·020 0·030 0·004 0·002

0·016 0·020 0·007 0·011 0·030 0·000 0·001

0·015 0·040 0·007 0·032 0·140 0·001 n.d.∗

0·002 0·105 0·020 0·106 0·110 0·010 0·020

0·0009 0·0001 0·0004 n.d.∗ 0·0001 — —

24 26 28d 29e

Values are lmol/mL. Values are mg P/(min×mL). c IP6–IP3: inositol hexa-, penta-, tetra- and triphosphate. d The second wet steep excluded and the first wet steep prolonged to 5 h and the dry steep prolonged to 17 h. e 3·2 volumes of lactic acid were used in the process. ∗ n.d.: not detectable. a

b

Phytase activitya

14

Phytase activity Experiment 7 12 17 20 23

A Ib

B IIb

A IIb

BDb

DPb

0·014 0·006 0·015 0·015 0·018

0·008 0·004 0·011 0·010 0·012

0·004 0·003 0·003 0·007 0·006

0·004 0·003 0·002 0·004 0·003

0·003 0·002 0·002 0·004 0·002

a

Measured indirectly as released phosphorus during incubation. Values are mg P/(min×g dry matter). b After the first wet steep (A I), before the second wet steep (B II), after the second wet steep (A II), before drying (BD) and dried end product (DP).

myo-inositol in the end product from Experiment 27 (1·29 vs 3·80 lmol/g d.m. phytate and 2·45 vs 2·68 lmol/g d.m. free myo-inositol). Thus, one wet steep not only gave a slightly higher level of free myo-inositol but also a higher phytate content in the end product. Myo-inositol is a small and water soluble compound which can leach to the steep water (Table V). Hence, the smaller amount of free myo-inositol in Experiment 26 compared to Experiment 27 can be explained by leakage of myo-inositol into the second steep water. In Experiment 28, where one prolonged wet steep step was used, the level of free myo-inositol in the steep water was 5–55 times higher than in the first wet steep water from Experiments 24, 26 and 29 (Table V). The amount of free myo-inositol in the steep water from Experiment 28 was 16% of the amount in the end product. The increase of free myo-inositol during the

IP6 (µmol/g d.m.)

Table VI

12

AI

B II

A II

BD

DP

21

37

10 8 6 4 2 0

0

1

6 7 Process time (h)

Figure 3 Phytate in samples taken out during the process after the first wet steep (A I), before the second wet steep (B II), after the second wet steep (A II) and before drying (BD). Process conditions for Experiments 1–3 were 55 °C during the whole process and 0·0, 0·1 and 0·6% (v/w) lactic acid solution concentration in both wet steeps, respectively, and for Experiments 4 and 5 they were 70 °C during the whole process and 0·1 and 0·6% (v/w) lactic acid solution concentration in both wet steeps, respectively. Ε, Experiment 1; Ε Φ, Experiment 2; Φ, Experiment 3; Φ Ε, Experiment 4; Φ, Experiment 5. Ε

process was not as big as one might expect considering how much phytate was degraded. The amount of inositol hexa-, penta-, tetra-, and triphosphate in the raw material was 14·8 lmol/g d.m. and the amount was 3·3 lmol/g d.m. in barley processed at optimal conditions for phytate degradation (T1=48 °C, T2=50 °C, C=0·8%, Experiment 25, Table II) while the increase of free myo-inositol in Experiment 25 was only 1·7 lmol/g d.m. We measured the content of myo-inositol mono- and diphosphates in Experiment 25 by using the method of Skoglund et al.34. The content

Hydrothermal processing of barley (cv. Blenheim)

of myo-inositol monophosphates was 0·9 lmol/g d.m. and the content of myo-inositol diphosphates was 1·1 lmol/g d.m. Hence, there was no major accumulation of lower myo-inositol phosphates. Thus, as once free myo-inositol has been released by hydrolysis of phytate during the hydrothermal process, only a minor part is stored in the seed as free myo-inositol while the major part is metabolised by the seed, which is in agreement with studies of germinated oats35 and dwarf beans36. As can be seen in Table VI there was a decrease of phytase activity, measured as released inorganic phosphorus during incubation at optimal conditions for phytase activity, in the barley seeds throughout the process, hence we could not produce an end product with high phytase activity. Our previous results24 show that phytate is degraded in whole grains of wheat, rye and oats in hydrothermal processes similar to that used in the current study. Optimisation of the present hydrothermal process for these cereals is now under investigation. However, optimal conditions for phytase activity in wheat13, rye37,38and oats14 differ from that of barley32 and optimal process conditions for phytate reduction during the hydrothermal process will probably also differ for the various cereals. We conclude that the hydrothermal process described can be used to achieve barley (cv. Blenheim) with a low phytate level and a high content of free myo-inositol, which can be used in the manufacturing of cereal based infant formulae and children’s food products with high bioavailability of minerals and myo-inositol. Other practical applications are, for example, gruels, flakes and muesli products.

4.

5.

6.

7.

8.

9.

10.

11.

12.

13. 14.

Acknowledgements

15.

This study was financially supported by the Nordic Industrial Foundation, Oy Lahden Polttimo AB, Semper AB, SL Foundation and Nutek.

16. 17.

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