The importance of lactic acid bacteria for phytate degradation during cereal dough fermentation

Lactic acid fermentation of cereal flours resulted in a 100 (rye), 95-100 (wheat), and 39-47% (oat) reduction in phytate content within 24 h. The extent of phytate degradation was shown to be independent from the lactic acid bacteria strain used for fermentation. However, phytate degradation during cereal dough fermentation was positively correlated with endogenous plant phytase activity (rye, 6750 mU g(-1); wheat, 2930 mU g(-1); and oat, 23 mU g(-1)), and heat inactivation of the endogenous cereal phytases prior to lactic acid fermentation resulted in a complete loss of phytate degradation. Phytate degradation was restored after addition of a purified phytase to the liquid dough. Incubation of the cereal flours in buffered solutions resulted in a pH-dependent phytate degradation. The optimum of phytate degradation was shown to be around pH 5.5. Studies on phytase production of 50 lactic acid bacteria strains, previously isolated from sourdoughs, did not result in a significant production of intra- as well as extracellular phytase activity. Therefore, lactic acid bacteria do not participate directly in phytate degradation but provide favorable conditions for the endogenous cereal phytase activity by lowering the pH value.     

Optimal conditions for phytate degradation, estimation of phytase activity, and localization of phytate in barley (cv. Blenheim)

Using a multivariate experimental design, optimal conditions for phytate degradation were found to be pH 4.8 and 57 degrees C in barley flour (cv. Blenheim) and pH 5.2 and 47 degrees C in a crude extracted phytase from barley. Three methods for measuring phytase activity in raw and hydrothermally processed barley were compared. Incubation at pH 5 and 55 degrees C for 60 min did not give significantly different results (p > 0.05), whereas incubation at pH 5 and 50 degrees C for 10, 20, 30, and 60 min gave significantly different results (p < 0.001) between methods. The change in microstructure of phytate globoids during hydrothermal processing showed that the degradation was highest in the scutellum cells and less in the aleurone layer.     

Treating Thin Stillage and Condensed Distillers Solubles with Phytase for Production of Low‐Phytate Coproducts

Fuel ethanol production from grains is mainly based on dry‐grind processing, during which phytate is concentrated about threefold in distillers dried grains with solubles (DDGS), a major coproduct. To reduce phytate in DDGS, Natuphos and Ronozyme industrial phytase preparations were used to treat commercially made thin stillage (TS). Changes in phosphorous (P) profile were monitored, and effects of reaction temperature, time, and enzyme concentration were investigated. Results showed that at a temperature ≤60°C for Natuphos phytase (≤70°C for Ronozyme phytase) and a concentration ≤4.8 FTU/mL of TS for Natuphos phytase (≤48 FYT/mL for Ronozyme phytase), a complete phytate hydrolysis (phytate P decreased to 0) could be achieved within 5–60 min of enzymatic treatment. Reduction in phytate P was generally accompanied by increase in inorganic P, whereas total P remained relatively unchanged. When condensed distillers solubles (CDS), the concentrated form of TS, was used as the substrate, phytate hydrolysis by each of the two enzyme preparations was as effective as on TS. Because a previous study from the author's laboratory showed that all types of P are mostly concentrated in TS and CDS but much less in distillers wet grains, phytase treatments of TS and CDS described in the present study can be an effective means in producing low‐phytate DDGS.     

Phytase and acid phosphatase activities in plant feedstuffs

A total of 183 samples representing 24 feedstuffs were analyzed for total phosphorus, phytate phosphorus content, phytase (Phy), and acid phosphatase (AcPh) activities with the objective to predict the capacity to hydrolyze phytic acid and to contribute to formulating environmentally adequate diets for monogastric animals. Of the cereals and cereal byproducts analyzed, only rye (5147 U kg(-)(1); 21 955 U g(-)(1)), wheat (1637 U kg(-)(1); 10 252 U g(-)(1)), rye bran (7339 U kg(-)(1); 56 722 U g(-)(1)), and wheat bran (4624 U kg(-)(1); 14 106 U g(-)(1)) were rich in Phy and AcPh activities. Legume seeds and oilseeds contained negligible Phy activity and a moderate amount of AcPh activity, except for kidney bean (33 433 U g(-)(1)) and full-fat linseed meal (13 263 U g(-)(1)). On the other hand, a significant linear regression between phytate phosphorus (y) and total phosphorus (x) was observed in cereal byproducts (R(2) = 0. 95; y = 0.8458x - 0.0367; P < 0.001) and oil seeds (R(2) = 0.95; y = 0.945x - 0.20; P < 0.001). Phy and AcPh were positively correlated with respect to phytate phosphorus in cereals, cereal byproducts, and other byproducts and negatively correlated in legume seeds and oilseeds. Except for cereals, the highest correlation between enzyme activities and phytate phosphorus was found for phytase. It is not possible to predict Phy and AcPh activities from phytate phosphorus content by linear and quadratic regressions. Finally, only highly significant and positive correlation was found between Phy and AcPh activities for cereals, cereal byproducts, and oilseeds.     

Oxidation of polyphenols in phytate-reduced high-tannin cereals: effect on different phenolic groups and on in vitro accessible iron.

After reduction of phytate with phytase, water slurries of two high-tannin cereal flours were incubated with polyphenol oxidase (mushroom tyrosinase), and the effects on different phenolic groups and on in vitro accessible iron were studied. Enzyme incubation was also performed after cooking, soaking, and germination of the cereals. Phytase incubation significantly decreased the phytate content, and incubation with polyphenol oxidase had a reducing effect on the total phenol content, as well as on the amount of catechol and resorcinol groups. The in vitro accessible iron increased when the cereals were incubated with phytase and polyphenol oxidase, and the highest accessibility of iron was obtained when the germinated samples were incubated. The results from this study imply that oxidation of polyphenols in high-tannin cereals, after reduction of phytate, may be used to increase the bioavailability of iron in foods prepared from these cereals.     

Soil–Phosphorus Mobilization Potential of Phytate Mineralizing Fungi

Four most efficient phytase and phosphatase producing fungi belonging to genera Aspergillus, Trichoderma, and Penicillium were isolated from the rhizosphere soil of leguminous, cereal, and vegetable crops. Efficacy order of fungi in terms of phytate hydrolysis under laboratory conditions was Aspergillus > Penicillium > Trichoderma. The test fungi released more of extracellular (E) phytase than intracellular (I) phytase (E: I- 3.44 - 6.03:1) and produced acid phosphatase activity ranging from 367- 830 μmol pNP ml^?1 h^?1. Aspergillus niger possessed the twin ability of phosphate mineralization and solubilization. The incubation studies in compost-amended soil exhibited the higher competence of Penicillium chrysogenum to improve the soil available P and increase the level of extractable organic P under alkaline soil to benefit P nutrition. Developing microbial inoculant using P. chrysogenum strain and its subsequent application to soil may help the marginal farmer to replenish soil P more economically compared to chemical fertilizer.     

Quantification of inositol phosphates using (31)P nuclear magnetic resonance spectroscopy in animal nutrition

A (31)P NMR method for quantitative determination of inositol phosphates in simple incubation samples of sodium phytate and Aspergillus niger phytase and in different types of complex samples, such as diets, digesta, and feces, is described. The inositol phosphates in complex samples were extracted with HCl, concentrated, and purified using freeze-drying and filtration and subsequently determined at pH 12.6 in aqueous solution using a (31)P NMR method. The (31)P NMR technique has as its main advantages over the HPLC techniques that it does not necessitate standards that may cause background matrix effects and that the spectra of inositol phosphates and orthophosphate appear in the same run without further sampling errors. The results of inositol hexaphosphate analysis with HPLC can be confirmed by this (31)P NMR method. Contents of inositol tetra-, tri-, di-, and monophosphate in the biological samples appear to be quantitatively not important. The (31)P NMR method can be applied for use in animal nutrition in general and studies of using phytase in diets for farm animals in particular, by measuring the content of inositol phosphates in feed ingredients, complete feeds, ileal contents, and feces of pigs and poultry.     

Moderate decrease of pH by sourdough fermentation is sufficient to reduce phytate content of whole wheat flour through endogenous phytase activity

Whole wheat bread is an important source of minerals but also contains considerable amounts of phytic acid, which is known to impair their absorption. An in vitro trial was performed to assess the effect of a moderate drop of the dough pH (around 5.5) by way of sourdough fermentation or by exogenous organic acid addition on phytate hydrolysis. It was shown that a slight acidification of the dough (pH 5.5) with either sourdough or lactic acid addition allowed a significant phytate breakdown (70% of the initial flour content compared to 40% without any leavening agent or acidification). This result highlights the predominance of wheat phytase activity over sourdough microflora phytase activity during moderate sourdough fermentation and shows that a slight drop of the pH (pH value around 5.5) is sufficient to reduce significantly the phytate content of a wholemeal flour. Mg "bioaccessibility"of whole wheat dough was improved by direct solubilization of the cation and by phytate hydrolysis.     

Microbial phytase activity and their role in organic P mineralization

Plants respond to their external environment to optimize their nutrition and production potential to minimize the food security issues and support sustainable agriculture system. Phosphorus (P) is an important nutrient for plants and is involved in plant metabolic processes. It is mostly available as orthophosphate and has a tendency to form complexes with cations. It has low mobility in soil, thus becoming unavailable for plant uptake that causes a reduction in plant growth and yield. Besides free P, phytate is the major form of organic P in soil and plant tissues. Phytases obtained from different sources, that is, plants, animals, and microorganisms, catalyze the hydrolysis of phytate and release available forms of inorganic P. The knowledge of mechanisms involved in catalytic activity of phytase obtained from microorganisms in soil is limited. This review summarizes the role of microbial phytase in releasing organic P by hydrolysis of phytate and factors affecting its activity in the soil.     

Effects of copper source and concentration on in vitro phytate phosphorus hydrolysis by phytase

Five copper (Cu) sources were studied at pH 2.5, 5.5, and 6.5 to determine how Cu affects phytate phosphorus (PP) hydrolysis by phytase at concentrations up to 500 mg/kg diet (60 min, 40-41 degrees C). Subsequently, Cu solubility with and without sodium phytate was measured. Adding Cu inhibited PP hydrolysis at pH 5.5 and pH 6.5 (P < 0.05). This inhibition was greater with higher concentrations of Cu. Tri-basic copper chloride and copper lysinate inhibited PP hydrolysis much less than copper sulfate pentahydrate, copper chloride, and copper citrate (P < 0.05). A strong negative relationship was observed between PP hydrolysis and soluble Cu at pH 5.5 (r = -0.76, P < 0.0001) and 6.5 (r = -0.54, P < 0.0001). In conclusion, pH, Cu concentration, and source influenced PP hydrolysis by phytase in vitro and were related to the amount of soluble Cu and the formation of insoluble copper-phytin complexes.     

Evaluation of antioxidant capacity of cereal brans

Several oat brans (crunchy oat bran, oat bran alone, and oat breakfast cereal) and wheat brans (wheat bran alone, wheat bran powder, wheat bran with malt flavor, bran breakfast cereal, tablet of bran, and tablet of bran with cellulose) used as dietary fiber supplements by consumers were evaluated as alternative antioxidant sources (i) in the normal human consumer, preventing disease and promoting health, and (ii) in food processing, preserving oxidative alterations. Products containing wheat bran exhibited higher peroxyl radical scavenging effectiveness than those with oat bran. Wheat bran powder was the best hydroxyl radical (OH*) scavenger. In terms of hydrogen peroxide (H2O2) scavenging, wheat bran alone was the most effective, while crunchy oat bran, oat bran alone, and oat breakfast cereal did not scavenge H2O2. The shelf life of fats (obtained by the Rancimat method for butter) increased most in the presence of crunchy oat bran. When the antioxidant activity during 28 days of storage was measured by the linoleic acid assay, all of the oat and wheat bran samples analyzed showed very good antioxidant activities. The Trolox equivalent antioxidant capacity (TEAC) assay was used to provide a ranking order of antioxidant activity. The wheat bran results for TEAC (6 min), in decreasing order, were wheat bran powder > wheat bran with malt flavor > or = wheat bran alone > or = bran breakfast cereal > tablet of bran > tablet of bran with cellulose. The products made with oat bran showed lower TEAC values. In general, avenanthramide showed a higher antioxidant level than each of the following typical cereal components: ferulic acid, gentisic acid, p-hydroxybenzoic acid, protocatechuic acid, syringic acid, vanillic acid, vanillin, and phytic acid.     

In vitro digestion characteristics of unprocessed and processed whole grains and their components

Chemical composition and in vitro digestion properties of select whole grains, before and after processing, and their components were measured. Substrates included barley, corn, oat, rice, and wheat. In addition to whole grain flours, processed substrates also were tested as were corn bran, oat bran, wheat bran, and wheat germ. Processing of most substrates resulted in higher dry matter and digestible starch and lower resistant starch concentrations. Dietary fiber fractions varied among substrates with processing. Digestion profiles for most substrates correlated well with their chemical composition. Corn bran and rice substrates were the least fermentable. Extrusion rendered barley, corn, and wheat more hydrolytically digestible and barley and oat more fermentatively digestible. Except for corn bran, all components had greater or equal fermentability compared with their native whole grains. Understanding digestion characteristics of whole grains and their components will allow for more accurate utilization of these ingredients in food systems.     

Orthophosphate, phytate, and total phosphorus determination in cereals by flow injection analysis

A flow injection spectrophotometric procedure with enzymatic hydrolysis was developed for determination of orthophosphate, phytate and total phosphorus in cereal samples. Phosphorus species were extracted from cereals with 0.05 mol L(-1) potassium hydrogen phthalate buffer solution at pH 5.7. Orthophosphate was directly determined in the extracts by molybdenum blue spectrophotometric method. The phytate was hydrolyzed by the enzyme phytase coupled to a solid phase packed into an enzymatic reactor, and the resulting hydrolyzed orthophosphate was also determined by spectrophotometry at 650 nm. After optimization for phosphorus species extraction and enzymatic hydrolysis, a linear calibration graph was obtained up to 196 x 10(-6) mol L(-1) orthophosphate (P conc = -2.67 + 0.52x, r = 0.9998). Measurements are characterized by relative standard deviation of 1.6% for a standard of 72 x 10(-6) mol L(-1) orthophosphate and no baseline drift was observed during 4 h operation periods. It provides 72 measurements per hour, with 2.4 x 10(-)6) mol L(-1) and 7.9 x 10(-6) mol L(-1) as detection and quantification limits, respectively.     

Quantification of a broad spectrum of lignans in cereals, oilseeds, and nuts

Twenty-four plant lignans were analyzed by high-performance liquid chromatography-tandem mass spectrometry in bran extracts of 16 cereal species, in four nut species, and in two oilseed species (sesame seeds and linseeds). Eighteen of these were lignans previously unidentified in these species, and of these, 16 were identified in the analyzed samples. Four different extraction methods were applied as follows: alkaline extraction, mild acid extraction, a combination of alkaline and mild acid extraction, or accelerated solvent extraction. The extraction method was of great importance for the lignan yield. 7-Hydroxymatairesinol, which has not previously been detected in cereals because of destructive extraction methods, was the dominant lignan in wheat, triticale, oat, barley, millet, corn bran, and amaranth whole grain. Syringaresinol was the other dominant cereal lignan. Wheat and rye bran had the highest lignan content of all cereals; however, linseeds and sesame seeds were by far the most lignan-rich of the studied species.     

Optimization and validation of the reversed-phase high-performance liquid chromatography with fluorescence detection method for the separation of tocopherol and tocotrienol isomers in cereals, employing a novel sorbent material

The separation and determination of tocopherols (Ts) and tocotrienols (T3s) by reversed-phase high-performance liquid chromatography with fluorescence detection has been developed and validated after optimization of various chromatographic conditions and other experimental parameters. Analytes were separated on a PerfectSil Target ODS-3 (250 × 4.6 mm, 3 μm) column filled with a novel sorbent material of ultrapure silica gel. The separation of Ts and T3s was optimized in terms of mobile-phase composition and column temperature on the basis of the best compromise among efficiency, resolution, and analysis time. Using a gradient elution of mobile phase composed of isopropanol/water and 7 °C column temperature, a satisfactory resolution was achieved within 62 min. For the quantitative determination, α-T acetate (50 μg/mL) was used as the internal standard. Detection limits ranged from 0.27 μg/mL (γ-T) to 0.76 μg/mL (γ-T3). The validation of the method was examined performing intraday (n = 5) and interday (n = 3) assays and was found to be satisfactory, with high accuracy and precision results. Solid-phase extraction provided high relative extraction recoveries from cereal samples: 87.0% for γ-T3 and 115.5% for δ-T. The method was successfully applied to cereals, such as durum wheat, bread wheat, rice, barley, oat, rye, and corn.     

Normal phase high-performance liquid chromatography method for the determination of tocopherols and tocotrienols in cereals

The eight vitamers of vitamin E (alpha-, beta-, gamma-, and delta-tocopherols and -tocotrienols) have different antioxidant and biological activities and have different distributions in foods. Some cereals, especially oat, rye, and barley, are good sources of tocotrienols. A fast procedure for the determination of tocopherols and tocotrienols (tocols) in cereal foods was developed. It involves sample saponification and extraction followed by normal phase high-performance liquid chromatography (HPLC). The results have been compared with those found by direct extraction without saponification. The method is sensitive and selective enough to be tested on a wide variety of cereal samples. The highest tocol levels were found in soft wheat and barley ( approximately 75 mg/kg of dry weight). beta-Tocotrienol is the main vitamer found in hulled and dehulled wheats (from 33 to 43 mg/kg of dry weight), gamma-tocopherol predominates in maize (45 mg/kg of dry weight) ), and alpha-tocotrienol predominates in oat and barley (56 and 40 mg/kg of dry weight, respectively).     

In Vitro Binding of Bile Acids by Rice Bran,Oat Bran,Wheat Bran,and Corn Bran

The in vitro bile acid binding by rice, oat, wheat, and corn brans was determined using a mixture of bile acids normally secreted in human bile at a physiological pH of 6.3. The objective of the study was to relate bile acid binding of cereal brans to health promoting properties. Three experiments were conducted testing substrates on an equal weight (dry matter) basis, an equal total dietary fiber (TDF) basis, and an equal TDF and equal fat basis. Each experiment was repeated to validate the results (for a total of six experiments). The relative in vitro bile acid binding of the cereal brans on an equal TDF basis considering cholestyramine as 100% bound was rice bran 51%, wheat bran 31%, oat bran 26%, and corn bran 5%. The data suggest that cholesterol lowering by rice bran appears to be related to bile acid binding. The primary mechanism of cholesterol lowering by oat bran may not be due to bile acid binding by soluble fiber. Bile acid binding did not appear to be proportional to the soluble fiber content of the cereal brans tested. Bile acid binding by wheat bran may contribute to cancer prevention and other healthful properties.     

Phytate degradation in a mixture of ground wheat and ground defatted soybeans during feed processing: effects of temperature, moisture level, and retention time in small- and medium-scale incubation systems

The optimal conditions for degradation of phytate (IP6, myo-inositol hexaphosphate) in a mixture of ground wheat and ground defatted soybeans (1:2, w/w) with added exogenous E. coli phytase were investigated at different temperatures (45, 60, 75, and 95 degrees C), moisture levels (25%, 35%, and 45%), and retention times (2-45 min). All treatment combinations were investigated in a small-scale mixer conditioner (experiment 1). The combined 45 degrees C and 45% moisture treatment was most efficient and reduced the content of IP6 by 86% during 45 min of incubation. This treatment combination was applied in a medium-scale mixer conditioner (experiment 2), and 76% reduction of IP6 at 45 min was obtained. During incubation, the content of lower groups of inositol phosphates, such as IP4 (myo-inositol tetraphosphate) and IP3 (myo-inositol triphosphate), increased significantly as the content of IP6 decreased. The major isomer formed was Ins(1,2,5,6)P(4).     

Changes in Phytate Content in Whole Meal Wheat Dough and Bread Fermented with Phytase‐Active Yeasts

The degradation of inositol hexakisphosphate (IP6) was evaluated in whole meal wheat dough fermented with baker's yeast without phytase activity, different strains of Saccharomyces cerevisiae (L1.12 or L6.06), or Pichia kudriavzevii with extracellular phytase activity to see if the degradation of IP6 in whole meal dough and the corresponding bread could be increased by fermentation with phytase‐active yeasts. The IP6 degradation was measured after the dough was mixed for 19 min, after the completion of fermentation, and in bread after baking. Around 60–70% of the initial value of IP6 in the flour (10.02 mg/g) was reduced in the dough already after mixing, and additionally 10–20% was reduced after fermentation. The highest degradation of IP6 was seen in dough fermented with the phytase‐active yeast strains S. cerevisiae L1.12 and P. kudriavzevii L3.04. Activity of wheat phytase in whole meal wheat dough seems to be the primary source of phytate degradation, and the degradation is considerably higher in this study with a mixing time of 19 min compared with earlier studies. The additional degradation of IP6 by phytase‐active yeasts was not related to their extracellular phytase activities, suggesting that phytases from the yeasts are inhibited differently. Therefore, the highest degradation of IP6 and expected highest mineral bioavailability in whole meal wheat bread can be achieved by use of a phytase‐active yeast strain with less inhibition. The strain S. cerevisiae L1.12 is suitable for this because it was the most effective yeast strain in reducing the amount of IP6 in dough during a short fermentation time.     

Determination of the activity of acidic phytate-degrading enzymes in cereal seeds

Five different methods were compared to elucidate the total activity of the acidic phytate-degrading enzymes present in the seeds of rye, wheat, and barley. Phytate-degrading activity was studied at pH 5.0 by quantifying the liberated phosphate. Rye showed the highest acid phytate-degrading activity among the cereals studied. Using an aqueous extract, only 30-50% of the activity was found (rye, 3443 mU g(-1) of grain; wheat, 1026 mU g(-1) of grain; barley, 1032 mU g(-1) of grain) compared to that found by the direct incubation of the dry-milled cereal grains in a buffered phytate-containing solution (rye, 6752 mU g(-1) of grain; wheat, 2931 mU g(-1) of grain; barley, 2093 mU g(-1) of grain). Extending the extraction time resulted in an increase in extractable phytate-degrading activity by, at maximum, 10-15%. Extraction of phytate-degrading activity is strongly enhanced in the presence of Triton X-100 and the protease inhibitor phenylmethylsulfonyl fluoride (rye, 6536 mU g(-1) of grain; wheat, 2873 mU g(-1) of grain; barley, 2023 mU g(-1) of grain), suggesting at least a partial association with membrane structures and a degradation by proteolytic activity during extraction. In addition, it was shown that determining phytate-degrading activity by quantification of the liberated inorganic phosphate is more robust and precise than determining phytate-degrading activity by quantification of the residual phytate.