Phytate Hydrolysis by Phytase in Cereals; Effects on In Vitro Estimation of Iron Availability

Phytate (inositol hexaphosphate) hydrolysis by endogenous and exogenous phytases was studied for their effect on increasing iron availability in cereals. Wheat bran and whole meal flours of rye and oats were soaked at optimal conditions for phytase activity (55°C, pH 5) for different time intervals. Phytate and its degradation products were determined by HPLC and related to iron solubility under simulated physiological conditions. Small amounts of phytate (< lμmol/g) had a strong negative effect on iron solubility. When inositol hexa- and pentaphosphates of wheat bran and rye flour were completely hydrolyzed by activating endogenous phytase, iron solubility under simulated physiological conditions increased from 3 to 53% (wheat) and 5 to 21% (rye). Addition of wheat phytase to uncooked oatmeal increased iron solubility from 4 to 11 and in precooked to 18%, while endogenous phytase of uncooked oatmeal had less effect on phytate digestion and iron solubility.     

Further studies on SS bonds in cereal glutelins

Flours of wheat, rye, barley, oats, maize and sorghum have been extracted to remove albumins, globulins and prolamins. The SS bonds of the starch-glutelin residues (4 to 8% protein) have been reduced with sulphite and titrated with phenyl mercury acetate (PMA) in the presence of 3 M -guanidine hydrochloride at 37 °C and, in its absence, at 2 and 37 °C. Attention has been paid to several possible sources of error including oxidation and the rate of diffusion of PMA into protein particles. Approximate values for the diffusion coefficients of PMA through cellulose and protein films were obtained. Titrations at 37 °C in the absence of guanidine hydrochloride are unsatisfactory due to reaction of intrachain SS links. Evidence in the literature suggests that the SS bonds titratable at 2 °C are inter-chain, possibly includingsomestrained intra-chain bonds. The results imply that most of the major polypeptide chains in the cereal glutelins examined, apart from barley and sorghum, contain two such bonds. In the case of barley glutelin probably less than half the chains have two labile SS bonds. Most of the chains in sorghum glutelin appear to have a single labile bond and the polymers may contain only a few chains. The molecular weights (in thousands) of the principal polypeptide chains of the glutelins, deduced from gel electrophoresis in sodium dodecyl sulphate (SDS), are: Cappelle-Desprez wheat 44, 41; Manitoba wheat, rye and barley, 44; oats, 33, 23; maize, 23, 19; sorghum, 22, 18.     

Phytate Reduction in Brown Beans (Phaseolus vulgaris L.)

Brown beans (Phaseolus vulgaris L.) were subjected to treatments to evaluate effects of pH, temperature, CaCl₂, tannase and fermentation on degradation of phytate. Soaking was performed at 21°C, 37°C and 55°C at pH 4.0, 6.0, 6.4, 7.0, and 8.0. Optimal conditions for phytate degradation were pH 7.0 and 55°C. After soaking 4, 8 or 17 hr at these conditions 79%, 87% and 98% of phytate was degraded, respectively. Addition of tannase enhanced reduction of phytate. Fermentation of presoaked whole beans resulted in reduction of 88% of phytate after 48 hr.     

Effect of sourdough processing and baking on the content of enniatins and beauvericin in wheat and rye bread

The occurrence of the emerging mycotoxins enniatins (ENNs) and beauvericin (BEA) has been reported in Fusarium-infected cereals. To study the effect of sourdough processing and baking on ENN B, ENN B1, and BEA concentrations, a recently developed stable isotope dilution assay for these mycotoxins was used. After milling of wheat and rye grains naturally contaminated with ENN B and ENN B1, approximately 70–82 % of the two ENNs were found in the bran fraction and the rest remained in flour. BEA was added to flour before sourdough fermentation. In an experiment on a microscale, dough was fermented for 24 h at 30 or 40 °C, which reduced part of the ENNs and BEA in particular at 40 °C. On a standard scale, mixing, resting, and proofing of the bread dough resulted in 13–19 % reduction of the ENNs compared with flour, but in no significant change of BEA. The final baking at 200 °C for 25 min led to a further decrease of the ENNs and BEA, ranging from 9 to 28 % compared with fermented dough. In case of rye sourdough bread, greater reductions of ENNs were found in crust than in crumb. For both wheat and rye flours, overall 25–41 % of ENN B, ENN B1, and BEA were reduced during the whole sourdough bread-making process.     

Phytate content and phytate degradation by endogenous phytase in pea (Pisum sativum)

In order to rapidly reduce the content of inositol tri–hexaphosphates in pea flour by action of the endogenous phytase, raw materials as well as incubation conditions have been evaluated. The phytate (inositol hexaphosphate) content was analysed in 27 pea varieties; the influence of storage time and the difference in phytate content between the germ and the cotyledon were determined. Furthermore, degradation of inositol phosphates by the endogenous phytase enzyme was studied in pea flour, germ and cotyledon. To find the maximum phytate degradation, the effects of temperature and pH during pea flour incubation were investigated. The most efficient phytate degradation in pea flour incubation was achieved at pH 7.5 and 45 °C. At this condition an almost complete degradation of phytate and a 66% reduction in the sum of inositol hexa‐, penta‐, tetra‐ and triphosphates were reached in 10 h. The storage time of pea seeds or removal of the germ did not have a major effect on the phytate content. Since several inositol pentaphosphate isomers were produced during phytate degradation, it can be concluded that peas contain several phytate‐degrading enzymes, or one phytate‐degrading enzyme with unspecific initial hydrolysation pattern. © 2001 Society of Chemical Industry.     

A comparison of the non-starch carbohydrates in cereal grains

The carbohydrates soluble in 80% ethanol and in water at 100°C were extracted from two varieties of wheat, barley, oats, rye, triticale, rice and malt. The highest concentrations of sugars soluble in 80% ethanol were found in malt and rye with the lowest in rice. Barley contained the most raffinose and rye contained the most fructan. Rye had the highest levels of water-soluble and total pentosans and barley the highest levels of water-soluble and total (1→3), (1→4)-β-glucans. The total arabinose:xylose ratio varied from 1.2 in wheat and triticale to 0.3 in oats. All were very low in uronic acids.     

Studies on the gluten-specific peptidase activity of germinated grains from different cereal species and cultivars

Grains of common wheat, spelt, emmer, einkorn, rye, barley, oats, and maize (two cultivars each) were germinated for 7?days at 15 and 25?°C, respectively, lyophilized, and milled into flour and bran. The rate of storage protein (prolamins, glutelins) degradation in the bran was determined by an extraction/HPLC method and was used as an indication for gluten-specific peptidase activity. Species and cultivars with high storage protein degradation during germination were selected for further studies. The peptidase activity of the germinated grains was determined by using two gluten substrates namely gliadin (wheat prolamin fraction) and the celiac-toxic peptide PQPQLPYPQPQLPY. The activity assay implied extraction of bran with a sodium acetate buffer (0.2?mol/L, pH 4.0), incubation of the extract with both substrates, and quantitation of gliadin or peptide degradation by RP-HPLC. The assays were simple and generated reproducible values for the peptidase activity. In general, the activity was strongly affected by cereal species, cultivar, germination temperature, and pH value of the application. Some of the bran extracts were capable of degrading both gliadin and the celiac-toxic peptide extensively. Maximum degradation rates of gliadin and the celiac-toxic peptide were 67 and 100?%, respectively. Alternatively, germinated cereal grains were kiln-dried at 45?°C for 24?h instead of being lyophilized and were separated into kernels and roots/sprouts. When compared to the lyophilized bran, the combined kiln-dried material showed a more balanced peptidase activity toward both the gliadin and the peptide substrate.     

Microbiological Method to Identify Irradiated Meat

Beef, chicken, mutton and pork exposed to -γ-radiation doses of 0–5 kGy were inoculated with Aeromonas hydrophila initially, after 7 days and 15 days of storage at 3°C and -11°C. After 18 hr incubation at 30°C TVBN values of nonirradiated samples were found to be 210 mg% and TVA value were 205 while the corresponding irradiated samples showed 30–80 mg% and 40–80. A similar pattern was observed in stored samples. A rapid method was developed by incubating chicken meat at 37°C for 6–7 hr with bacteria followed by estimation of volatile acids (TVA) and bases (TVBN). A 40–50% reduction in TVA and TVBN in irradiated samples was observed suggesting its possible application for detecting irradiated meat samples.     

Rye bread reduces plasma cholesterol levels in hypercholesterolaemic pigs when compared to wheat at similar dietary fibre level

BACKGROUND: Rye is a whole‐grain cereal with the potential of contributing to a healthy diet, but research on rye in relation to chronic diseases is scarce compared to wheat and oats. In this study, a total of 17 hypercholesterolaemic pigs were fed high‐fat high‐cholesterol rye (n = 9) or wheat‐based buns (n = 8) with similar dietary fibre (DF) content for 9–10 weeks to study the effect on cardiovascular risk factors. RESULTS: Ingestion of rye bread resulted in a 40% lower plasma total and LDL cholesterol compared to the wheat group, whereas HDL cholesterol, insulin and glucose concentrations were not affected by dietary treatments. Intestinal viscosity was 7.2 times higher, and organic matter and fat digestibility significantly reduced in the pigs fed rye buns. The hepatic expression of the cholesterol 7α‐hydroxylase gene (CYP7A1) was lower in rye‐fed pigs, whereas four other key genes involved in cholesterol metabolism were not affected. Plasma cholesterol correlated inversely with intestinal viscosity and organic matter digestibility. CONCLUSION: The ability of DF from rye to interfere with digestion and absorption is more important for whole‐body cholesterol homeostasis than regulation in the liver at gene level. Copyright © 2008 Society of Chemical Industry     

Steeping of Starch at Various Temperatures-Effects on Functional Properties

Functionality of wheat starches steeped at various time and temperature conditions was studied in bread, cake and pie filling systems. Treatment at 50°C reduced the bread volume. Crumb softness in breads increased with duration and temperature of the treatment. Addition of 3–10% (flour basis) treated starch (40°C for 3 days) reduced firmness of breads. Cakes with starches treated at 25°C and 40°C up to 3 days were acceptable; starch treatment at 50°C caused collapse of cakes. The consistencies of pie fillings are increased by starch treatment at 25°C. Starches treatment at higher temperatures (40–50°C) caused high initial consistencies of fillings which were reduced during storage.     

Determination of celiac disease-specific peptidase activity of germinated cereals

A method to determine the celiac disease-specific peptidase activity of different germinated cereals was developed. Kernels of common wheat, spelt, emmer, einkorn, rye, and barley were germinated, lyophilized, and milled into flour and bran. The latter was extracted at pH 4.0 to obtain a solution enriched with peptidases. The synthetic α-gliadin peptide with the amino acid sequence PQPQLPYPQPQLPY (peptide IV), which has been shown to be toxic for celiac disease patients, was selected as substrate for bran peptidases. It was quantified by reversed-phase high-performance liquid chromatography on C₁₈ silica gel. For kinetic studies, rye bran extract was incubated with peptide IV at 50 °C and pH 6.5. The peptide was degraded continuously, and only 30.2% of the original peptide was detected after 90 min. Accordingly, the bran extracts of all cereals were investigated. The incubation time was set to 60 min at 50 °C, and the degradation of peptide IV was performed at pH 4.0 and 6.5, respectively. Except for rye, peptide degradation was faster at pH 4.0 than at pH 6.5. At pH 4.0, emmer extract was most active, followed by spelt, common wheat, and einkorn extracts. The activity of rye and barley extracts was significantly lower. In conclusion, the method is easy to perform, quick, and provides reproducible results. It can be applied to other peptidase sources such as bacterial or fungal cultures to optimize peptidase preparations suitable for detoxifying gluten-containing food or for drugs to treat celiac disease.     

The Inhibitory Effects of Brans and Their Aqueous Extracts on the Lipolysis of Tributyrin Catalyzed by Calf Pregastric Lipase

The ability of red and white wheat brans and oat bran to inhibit calf pregastric lipase (CPGL)‐catalyzed hydrolysis of tributyrin has been studied in vitro at pH 6.5, 37 °C. Brans that had been presoaked in Bis‐Tris buffer at pH 6.5, 4 °C reduced CPGL activity by 13% to 20% after being soaked for 0.25 h and by 17% to 30% after being soaked for 24 h. Maximum reduction of CPGL activity was seen within 2 to 5 min incubation between coarse red wheat bran and CPGL. The results are discussed in terms of likely inhibitory components within the brans and their water‐soluble components and their roles in inhibiting pregastric and other lipase‐catalyzed hydrolysis of dietary lipids.     

Phytate content is reduced and β‐glucanase activity suppressed in malted barley steeped with lactic acid at high temperature

The effect of different steeping conditions on phytate, β‐glucan and vitamin E in barley during malting was studied by a full factorial experiment with three variables (steeping temperature, barley variety and steeping additions). Addition of lactic acid to the steeping water induced a reduction of phytate during steeping and germination, especially in combination with the high steeping temperature (48 °C). Furthermore, lactic acid and high temperature steeping inhibited β‐glucanase development, resulting in a well‐preserved β‐glucan content after germination. When steeping was conducted without addition of lactic acid, the low steeping temperature (15 °C) promoted development of both phytase and β‐glucanase activity during germination. A slightly higher level of tocopherols and tocotrienols was observed in samples steeped at 15 °C than in samples steeped at 48 °C. However, addition of lactic acid reduced the amount for both temperatures. When lactic acid bacteria were added to the steeping water none of the parameters studied differed from samples steeped with water only. The results show the possibility of combining phytate degradation with a preserved β‐glucan content during malting and can thus be of interest for development of cereal products with improved nutritional value. Copyright © 2004 Society of Chemical Industry     

The influence of germination conditions on beta-glucan, dietary fibre and phytate during the germination of oats and barley

This study aimed to quantify the changes caused by varying germination conditions on the contents of some bioactive compounds in barley and oats. Samples of the two grains were germinated at temperatures between 10 and 20 °C for a period of 2–6 days, using a two-dimensional central composite design. The germination temperature had only minor effect in comparison with the germination time. Slight changes in the mineral content of the malts were observed, mainly caused by steeping. Phytate has been seen as an anti-nutritional compound, as it complexes minerals and lowers their bioavailability. The phytate content in barley malts was considerably lower than in the native kernels. Variations in the germination conditions did not have a significant effect on phytate content. In oats, degradation of phytate was significantly enhanced by prolonging the germination period. It was possible to retain the amounts of soluble dietary fibre, when short germination periods were applied. However, long germination periods caused an extensive breakdown of soluble dietary fibre, especially beta-glucan. The content of insoluble fibre, however, was increased by applying long germination periods for oat malts.     

Interaction of phytate with protein and minerals in a soybean–maize meal blend depends on pH and calcium addition

In order to investigate possible interactions of phytate with protein and minerals in simplified animal diets, studies were conducted on the solubility of endogenous phytate, protein and essential minerals in a soybean–maize meal blend within a physiological relevant pH range. The blend was mixed with water for 10 min and then allowed to incubate at 40 °C (30 min) after adjustment of the pH. Finally, soluble phytate, protein, zinc, manganese and iron were determined. Phytate and mineral solubility was highly influenced by pH whereas protein solubility was less affected. Addition of 5 g Ca²⁺ kg^?1 drastically reduced the solubility of phytate, zinc, manganese and iron at pH above 4.4, indicating that the formation of insoluble phytate–mineral complexes is increased in the presence of calcium. The action of pepsin increased the solubility of protein and phytate at pH below 4, indicating that insoluble phytate–protein complexes are present at low pH. Calcium had the same solubilising effect as pepsin at pH 2–4 but to a lesser degree. Copyright © 2007 Society of Chemical Industry     

Zinc and Phytate Distribution in Peas. Influence of Heat Treatment, Germination, pH, Substrate, and Phosphorus on Pea Phytate and Phytase

The largest proportions of zinc and phytate, 88.7 and 97.1%, respectively, were in the Garfield pea cotyledon; the greatest concentrations were in the germ. Cooking peas by two different methods resulted in 13% phytate reduction. Peas incubated 6.5 hr from 25 to 80°C yielded maximum phytate loss (25%) at 60°C due to phytase activated hydrolysis. Germination (10 d) decreased pea phytate 75% and increased phytase activity 12-fold. Semi-purified germinated pea phytase showed temperature optimum at 45°C, pH optimum of 5.2, 30% inhibition by 1 mM inorganic P, and substrate preference for pyrophosphate. Incubation of early germinated peas at optima pH and temperature is suggested for maximum phytate reduction.     

The Effect of Endogenous Pectinases on the Consistency of Tomato–Carrot Purée Mixes

Tomato and carrot were subjected to a split-stream process designed to produce a tomato–carrot suspension with reduced consistency. Raw tomatoes, containing pectinmethylesterase and endo-polygalacturonase, were mixed with thermally pretreated (blanched versus cooked) carrots containing different levels of solubilized pectin. After mixing the vegetables, tomato pectinases were shown to act on both tomato and carrot pectin in case an incubation step at medium temperature level (30 min, 40 °C), to allow enzyme action, was performed. Carrot pectin, when present in a mix of tomato and blanched (5 min, 95 °C) carrot, was solubilized as well as depolymerized, whereas depolymerization of the thermo-solubilized carrot pectin by the tomato pectinases was observed in the tomato–carrot purée containing cooked (30 min, 95 °C) carrots. The final serum pectin properties were however similar for both purée types. Carrot contributed more to the consistency of the purée mix compared with tomato but by stimulating the action of the tomato pectinases at mild temperature (30 min, 40 °C), this contribution was lost which resulted in a consistency reduction of the purée mix. This purée liquefaction was larger for the tomato–carrot purée containing blanched instead of cooked carrots. Based on the results, it is suggested that the liquefying effect is related to solubilization and degradation of pectin that is counteracted by a reduction in particle size. The purée mix containing cooked carrot showed in this respect smaller particle sizes than the mix containing blanched carrot.     

Effect of malt pretreatment on phytate and tannin level of two sorghum (Sorghum bicolor) cultivars

The seeds of two cultivars of Sudanese sorghum (Sorghum bicolor), namely Wad Ahmed and Tabat, were germinated for 4 days to obtain 1‐, 2‐ and 4‐day‐old malts. Sorghum malt (5% and 10%) was added to sorghum flour. The mixtures were incubated at 30 °C with shaking for 30, 60, 90 and 120 min. Malting loss was very slight for both cultivars and for all incubation periods. Phytic acid and tannin contents were assayed for all treatments. The results revealed that phytate and tannin contents were significantly (P ≤ 0.05) reduced when sorghum flour was pretreated with malt. When a mixture containing 10%, 4‐day‐old malt and sorghum flour was incubated for 120 min, it significantly (P ≤ 0.05) reduced phytate and tannin contents by 92% and 98%, respectively, for Wad Ahmed cultivar, while for Tabat they were reduced by 93% and 96%, respectively. The rate of reduction of phytate and tannin content increased with incubation time and malt age and concentration.     

Effects of processing and storage on walnut (Juglans regia L) tannins

Walnut tannins were maximally extracted with absolute methanol and an extraction time of 60 min. Storage (21 days at 25 °C) significantly reduced (20–40% reduction) the assayable tannin content in walnuts. The ranges of tannin content in freshly cracked inshell and shelled walnuts were respectively 372–1095 and 363–667 mg catechin equivalent per 100 g dry weight. The assayable tannin content in inshell and shelled walnuts was 10–20% higher when the particle size was reduced from 2‐ to 8‐mesh. Using 0.5 as compared to 2% (w/v) vanillin as colour development reagent yielded 15–20% higher assayable tannin contents. The assay colour development reached a maximum after 20 min of incubation at 25 °C. Roasting (204 °C for 5 min) caused a small (14%) but significant reduction in assayable tannins. Soaking in aqueous alkali solutions was more effective (44–100% reduction) than soaking in aqueous acid solutions (6–76% reduction) in decreasing the assayable tannin content in walnuts. Microwave heating in distilled deionised water resulted in 93–98% reduction in walnut assayable tannins. Blanching at 100 °C for 2 min was as effective as alkali soaking in significantly reducing the amount of extractable walnut tannins (98% reduction). The tannin content in blanched walnuts was positively related to astringency scores (r = 0.92). © 2001 Society of Chemical Industry     

Supplementation of alkaline phytase (Ds11) in whole-wheat bread reduces phytate content and improves mineral solubility

Abstract: In this study, alkaline phytase was added to whole‐wheat bread and the phytate content and mineral profiles were compared to commercially available acidic phytase. At neutral pH, some phytate (approximately 20%) was degraded by endogenous phytase in wheat flour, while 40% of phytate was hydrolyzed by alkaline phytase DS11 and a 35% reduction was observed with acidic phytase. Most of the enzymatic activity occurred during the proofing stage, and the rate of reaction depended on pH. DS11 phytase effectively degraded the phytate level within a 30 min treatment at pH 7; however, at least 60 min was needed with acidic phytase to achieve the same hydrolysis level. Mineral profiles were also dramatically affected by the phytate reduction. The biggest increase was observed in Fe²⁺ by the phytase treatment. The Fe²⁺ content increased 10‐fold at pH 7 and 8‐fold at pH 5 with alkaline phytase DS11. Alkaline phytase DS11 was shown to be effective at phytate reduction in whole‐wheat bread preparation. Additionally, phytate degradation enhanced the mineral availability of bread.