An optimised micro-titer plate method for characterisation of endogenous rye phytase under industrial rye bread making conditions

A modified rapid method for the determination of phytase activity has been developed by measurements of molybdenum blue complex using micro-titer plate reader. This method requires only a small amount of extract, and it is possible to make more replicates at the same time and thus a better sampling and a higher capacity compared to measurements on spectrophotometer, which is traditionally used for those measurements. The extraction time was reduced by 36% compared to standard method and the extinction coefficient for molybdenum blue complex was determined instead of using P-standard solutions. To demonstrate the new method it was applied to the determination of the activity of phytase at a range of pH values and temperatures relevant to the rye bread making process. The activity of rye phytase was 320 nkatal g⁻¹ grains (pH 5.5, 37 °C). The temperature optimum was 45–55 °C and the pH optimum 6.0. The study revealed that the rye phytase is very pH-sensitive and quite stable at different temperatures during the whole bread-making process. The enzyme activity of the endogenous rye phytase is optimal for a total degradation of phytic acid during rye bread making and thus the full bioavailability of phytate-bound minerals.     

Inositol Phosphates in Processed Foods

Myo-inositol bis-, tris-, tetrakis-, pentakis- and hexakisphosphates (IP2–6) were quantified in nine thermally processed or fermented food products. Total inositol phosphate content (mmol/kg dry weight) ranged from 1.35 in white bread to 23.26 in tofu and 26.05 in soy isolate. In all foods analyzed, inositol hexakisphosphate (phytic acid) accounted for more than 40% of the total inositol phosphates on a molar basis. Step-gradient ion exchange and ferric chloride precipitation methods for phytate determination were evaluated with a soy isolate sample to which inositol phosphates or adenosine triphosphate (ATP) had been added individually. Both methods measured all of the inositol phosphates, while the former also measured ATP.     

体外消化/Caco-2细胞模型分析发酵对面粉铁生物利用率的影响

中国居民膳食以谷物为主食,谷物中铁吸收率低是造成营养性贫血的主要原因,适当的加工方式可缓解谷物中植酸、多酚等物质对铁生物利用率(Fe bioavailability,FeBV)的影响。为考察酵母发酵对面粉FeBV的影响,采用体外消化/Caco-2细胞模型。结果表明,面团在发酵后pH值呈下降趋势,酸度呈上升趋势;多酚、植酸含量降低,植酸酶活性升高,以上各指标在发酵前后差异均具有统计学意义(P0.05)。发酵后面粉样品的FeBV增加约5%~38%,大多数面粉FeBV发酵前后比较,差异具有统计学意义(P0.05)。结论:发酵可降低面粉的pH值,增加酸度,有利于面粉多酚和植酸的降解以及植酸酶活力的升高,可有效提高面粉FeBV。     

The Influence of Soaking and Germination on the Phytase Activity and Phytic Acid Content of Grains and Seeds Potentially Useful for Complementary Feedin

ABSTRACT: Phytic acid, a potent inhibitor of mineral and trace element absorption, occurs in all cereal grains and legume seeds. The possibility to increase phytase activity and/or reduce the phytic acid content by soaking and germination was investigated in a wide range of grains and seeds, but not found to be effective. Germination, but not soaking, increased phytase activity 3 to 5-fold in some cereal grains and legume seeds, while the influence on phytic acid content was insignificant in most materials tested. High apparent phytase activity was found in untreated whole grain rye, wheat, triticale, buckwheat, and barley. Their usefulness as sources of phytase in complementary food production should be further investigated.     

植酸提高米曲霉酶活力的机理

米曲霉能分泌植酸酶,如制曲中添加一定量植酸,经植酸酶降解产生肌醇和磷酸,肌醇激活酶系,从而提高米曲霉的酶活力。（一平     

Effect of Fermentation Time on the Inositol Phosphates of Bread

Bread was made by an AACC procedure using whole wheat flour and 70%-extraction wheat flour. Five different fermentation times were applied. Six inositol phosphates and inorganic phosphate were separated, identified and quantified in the bread. It was found that phytate decreased and the inorganic phosphate increased, with the largest decrease in phytate occurring during first 30 min of fermentation. The content in intermediate inositol phosphates also changed and it was only after considering their phosphorus content that an overall phosphorus balance could be achieved.     

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.     

Preparation of High Bran Arabic Bread with Low Phytic Acid Content

Incubation of wheat bran at pH 5.2 and at a temperature of 55°C for various periods of time caused an increase in the levels of inorganic phosphorus and inositol phosphates and a simultaneous decrease in the level of phytic acid. When Arabic bread containing different levels of bran was prepared, up to 32% of the original phytic acid was lost during the process of bread preparation. Incorporation of wheat malt in the bread formula did not have any effect on phytic acid loss while incubation of bran in an acidic medium followed by repeated decantation of the incubation liquid produced a high bran bread almost free of phytic acid.     

Phytins?ure in Getreide und Getreideerzeugnissen

Zusammenfassung Phytinsäure in Lebensmitteln wird für eine verringerte Bioverfügbarkeit von essentiellen Mineralstoffen verantwortlich gemacht; sie kann während der Verarbeitung durch Phytase teilweise abgebaut werden. Der durchschnittliche Phytinsäuregehalt betrug in Roggen 8,18 mg/g und im Mehl der Type 997 3,44 mg/g; im Ganzkornmaterial wurden durchschnittlich Phytaseaktivitäten von 3,7 U/g und im Mehl 2,6. U/g gefunden. In den beiden Kornhälften (quergeteilt) waren Phytat und Phytase etwa gleichmäßig verteilt. Während der dreitägigen Keimung blieb die Phytaseaktivität konstant, der Phytinsäuregehalt nahm um ein Drittel ab. Bei der Vermahlung und dem Schälen von Roggen wurden die größten Mengen an Phytinsäure und Phytase mit Schrot- und Grießkleien bzw. Schälfraktionen entfernt. Es wird daraus geschlossen, daß Substrat und Enzym in den gleichen morphologischen Strukturen, wenn auch intrazellulär getrennt, vorkommen. Küchentechnische Zubereitungsformen von Vollkornprodukten waren für eine Phytatreduzierung um so wirkungsvoller, 1.) je feiner das Getreide vermahlen war, 2.) je mehr Wasser zugegeben wurde und 3.) je länger Phytase im optimalen Temperaturbereich einwirken konnte. Extrusion von Vollkornschrot bewirkte erst bei einer hohen Temperatur ( 170 °C) eine 23%ige Reduktion, während Phytase schon bei 80 °C um etwa 80% geschädigt wurde.

---

Phytic acid in cerealsI. Phytic acid and Phytase in rye and rye products

Summary Phytic acid in food is considered to be responsible for a reduced bioavailability of essential dietary minerals; its detrimental effects can be diminished by hydrolysis with phytase during processing. The average phytic acid content was 8.18 mg/g and 3.44 mg/g and average phytase activity was 3.7 U/g and 2.6 U/g in rye kernels and in flour (Type 997, 1.09 ash content), respectively. Phytate and Phytase were about equally distributed between the two kernel halves (cross sections). During the early stages of germination (3 days) phytase activity did not change, and phytic acid content was reduced to 67%. After milling most of the phytic acid and phytase activity were found in the bran fractions. It is concluded that substrate and enzyme are present in the same kernel structures but separate within the cells. Cooking of ground rye caused a phytate hydrolysis which was the more effective 1.) the smaller the particle sizes were, 2.) the more water was added, and 3.) the longer phytase worked at optimum temperature. Extrusion cooking of the rye whole flour at up to 100 °C did not influence the phytic acid level but caused a 23% reduction at 170 °C. Phytase activity was reduced by 80% by extrusion cooking at 80 °C.

---

---

Veröffentl.-Nr. 5342 der Bundesforschungsanstalt für Getreide-und Kartoffelverarbeitung, Detmold     

Comparative study of colorectal health related compounds in different types of bread: Analysis of bread samples pre and post digestion in a batch fermentation model of the human intestine

Seven different types of wheat and rye bread were analysed for colorectal health related compounds, pre and post digestion, in batch fermentation model of the human intestine. Pre digestion, higher amounts of colorectal health-related dietary fibre compounds (soluble/insoluble/total dietary fibre, arabinoxylans, β-glucans) and phytochemicals (mono-/di-phenolic acids, phytic acid, hydroxymethylfurfural) were detected in wholemeal than in refined flour types of bread, as well as in rye flour types than in wheat flour types of bread. Post digestion, faecal bacterial metabolites of colorectal health promoting (acetate/propionate/butyrate, lactate, free mono-/di-phenolic acids) and impairing (amino metabolites, bile acid metabolites) activities were found in fermentation supernatants of bread samples. All types of bread positively affected faecal bacterial metabolism; among the different types of bread, the highest stimulation of organic acid production (acetate/propionate/butyrate, lactate) and the lowest detrimental bacterial enzyme activities (β-glucuronidase, urease) were detected for wheat flour bread, whereas the strongest retardation of bacterial bile acid degradation and the strongest stimulation of phenolic acid metabolite release (phenylpropionic/phenylpropenoic acid derivatives) were induced by wholemeal rye bread. This study for the first time presents a qualitative and quantitative overview over the broad spectrum of colorectal health related compounds in high- and low-fibre types of bread, pre and post in vitro digestion, and highlights the significance of bread for the preventive nutritional intervention of colorectal cancer.     

The analysis for inositol phosphate forms in feed ingredients

Limited data are available on the content of inositol phosphates in feed ingredients. The doubtfulness over the identity and literature values of the inositol hexaphosphate (phytate) analysed in feed ingredients by the classical iron precipitation method prompted the analysis for inositol phosphates in some feed ingredients using a modified high-performance liquid chromatography method. The inositol phosphates analysed were inositol triphosphate (IP3), inositol tetraphosphate (IP4), inositol pentaphosphate (IP5) and inositol hexaphosphate (IP6). At least 97% of the inositol phosphates in grains occurred as IP6 and the remainder as IP5. In milled products the relative percentage of IP5 was higher (up to 14·5% in wheat bran) but the overall IP6 relative percentage was still high (more than 82%). IP3 and IP4 were not present in grains, and their amounts were very low when present in their milling or processed fractions. The absolute IP6 content was highest in rice bran and wheat bran at 68·31 and 47·63 g kg⁻¹, respectively. The results indicate that processed plant products may contain hydrolysis products of IP5 and IP6. These products contain greater percentages of IP3, IP4 and IP5 than in grains or milling products. © 1998 SCI.     

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.     

Effect of Flour Extraction Rate and Baking on Thiamine and Riboflavin Content and Antioxidant Capacity of Traditional Rye Bread

ABSTRACT:  The effect of rye flour extraction rates and baking on thiamine and riboflavin content, and antioxidant capacity of traditional rye bread were studied and compared with white wheat flour. The content of thiamine was higher (10.9%) in rye dough formulated with dark rye flour (F-100%; extraction rate of 100%) than in rye dough formulated with brown rye flour (F-92%; extraction rate of 92%) that was similar to dough made with wheat flour. The riboflavin content in rye dough made from flour F-100% was also higher (16%) than in dough formulated with flour F-92%, and both provided larger riboflavin content than wheat dough. Baking led to reductions in thiamine of 56% for wheat bread and of 20% for both rye breads; however, this process caused only a 10% decrease in riboflavin for wheat bread and a 30% decrease for rye breads. Trolox equivalent antioxidant capacity, peroxyl radical scavenging capacity, DPPH radical scavenging activity, and Folin–Ciocalteu reducing capacity were higher in rye than in wheat dough and bread. Baking process produced slight changes in antioxidant activity, except for Superoxide Dismutase-like activity where a sharp decrease was observed. Our findings showed that rye breads are an important source of B vitamins and rye breads formulated with dark and brown flours showed better antioxidant properties than wheat bread. Therefore, rye breads should be more widely recommended in human nutrition.     

Phytic acid concentration in selected raw materials and analysis of its hydrolysis rate with the use of microbial phytases during the mashing process

Phytic acid present in the raw materials can complex with many compounds and therefore limit their availability to the yeast during the alcoholic fermentation process. An effective utilization of biogenic compounds bound in phytates requires a detailed analysis of the raw materials for their phytic acid content. The aim of this study was to characterize the major technological parameters for selected raw materials used in the distilling industry (maize, rye, wheat and triticale grain) and to determine the phytic acid content and the IP6/total phosphorus ratio. The phytic acid hydrolysis rate during the mashing process, with the use of microbial phytases, was analysed. The highest phytic acid concentrations (2.30 ± 0.20 mg/g dry matter) and the highest IP6/total P (80.42 ± 6.99%) were observed in the maize grain samples. Therefore, further studies on the phytic acid hydrolysis rate with the use of various phytases were conducted for the maize grain. The highest hydrolytic activity was observed for the Phytase 10000L preparation. This was the preparation that hydrolysed the phytic acid completely in up to 90 min. The application of a highly effective phytase, in ethanol production from maize grain, could lead to a more effective utilization of the biogenic compounds during the fermentation process. Copyright © 2015 The Institute of Brewing & Distilling     

Selection of lactic acid bacteria with high phytate degrading activity for application in whole wheat breadmaking

Whole wheat products although highly recommended from the nutritional point of view, contain high levels of phytic acid, an antinutrient that decrease the mineral bioavailability. The objective of this study was to select strains with high phytate-degrading activity from different parts of the gastrointestinal tract of chickens following a phytate-rich diet and to test their suitability for the breadmaking process of whole wheat bread. Different lactobacilli and bifidobacteria strains were isolated and individually assayed for phosphatase and phytase activities, since both enzymes could contribute to the degradation of phytate. The isolates showing the highest phytate degrading activity belonged to the species Bifidobacterium dentium, Lactobacillus reuteri (L-M15) and Lactobacillus salivarius (L-ID15). The two lactobacilli L-M15 and L-ID15 were selected and tested their fermentative ability in whole wheat breadmaking. Whole wheat breads in the presence of the selected lactobacilli had similar technological quality than the control (in absence of lactobacilli) and extended freshness; moreover, their presence resulted in bread crumbs with lower levels of inositol phosphates. Overall, the two intestinal lactobacilli strains showing high phytate degrading activity were proven to have good properties for being used as starters in whole wheat breadmaking process.     

Effects of yeast and bran on phytate degradation and minerals in rice bread

ABSTRACT:  Rice bread is a potential alternative to wheat bread for gluten-sensitive individuals. Incorporation of rice bran into bread made from white rice flour adds flavor but also phytic acid, which can reduce the bioavailability of minerals. Breads with varied amounts of defatted bran and yeast were prepared to determine their effects on the phytate and mineral contents of the bread. A completely randomized factorial design was used with bran levels of 3.7%, 7.3%, and 10.5% of the dry ingredients and yeast levels of 1.6%, 3.2%, and 4.7%. Increasing the amount of bran decreased the phytate degradation from 42% at the lowest level of bran to 10% at the highest, and the amount of yeast had no significant effect. The bran contributed substantial amounts of magnesium, iron, and zinc. Breads with the lowest level of bran had phytate-to-zinc molar ratios between 5 and 10, which suggest medium zinc bioavailability. Rice bread is a tasty and nutritious food that is a good dietary source of minerals for people who cannot tolerate wheat bread.     

Monitoring the stepwise phytate degradation in the upper gastrointestinal tract of pigs

The degradation and formation of inositol phosphates as affected by microbial phytase and gastrointestinal enzyme activities during the passage of phytate through the stomach and small intestine were studied in two experiments with four barrows and three collection periods. The degradation and formation of inositol phosphates were measured at the duodenal and ileal sites using Cr‐NDR, TiO₂ and Co‐EDTA as indigestible markers. In experiment 1, the effect of graded doses of Aspergillus niger phytase (0, 150 and 900 FTU Natuphos^® kg^?1), added to a maize–soybean meal‐based diet with very low intrinsic phytase activity on the degradation of phytate and the formation of inositol phosphates during digestion in the stomach and small intestine was investigated. In experiment 2, three different mixtures of inositol phosphates, produced by Aspergillus niger phytase, containing mainly high, intermediate and low phosphorylated inositol phosphates, were added to the same maize–soybean meal‐based diet as used in experiment I. The fate of the inositol phosphates during digestion in the stomach and small intestine was studied. Experiment 1 showed that the extent of phytate degradation was dependent of the graded dietary phytase activities. At high phytase activity (900 FTU kg^?1 of diet), strong phytate degradation occurred and the once hydrolysed phytate was rapidly dephosphorylated to lower inositol phosphates (mainly inositol di‐ and triphosphates). Intermediate inositol phosphates, such as inositol tetraphosphates, were quantitatively unimportant in duodenal and ileal digesta. At a phytase activity of 150 FTU kg^?1 of diet, a broader spectrum of intermediate inositol phosphates was determined, which was probably due to a slower breakdown of phytate. Experiment 2 showed as a predominant result that lower inositol phosphates such InsP₄ and InsP₃ were degraded, whereas InsP₂ accumulated in the duodenal and ileal digesta. No substantial disappearance of phytate from the stomach and small intestine was found when high concentrations of soluble phytate were added to the diet, which indicates that no substantial phytate absorption occurs in the upper part of the pig gut. Copyright © 2005 Society of Chemical Industry     

Effect of Milling Extraction on the Inositol Phosphates of Wheat Flour and Bread

SRW wheat was experimentally milled to 70%, 80%, 90% and 100% extraction flours, and “standard” bread was prepared from these flours. Six inositol phosphates and inorganic phosphate were found in all flours and breads. Inositol hexaphosphate, inorganic phosphate and total P increased with the degree of extraction. About 2/3 of the inositol hexaphosphate disappeared as a result of bread making, but an overall phosphate balance was possible only after taking into account the intermediate inositol phosphates. Commercial white bread and whole wheat bread were also analyzed for the entire spectrum of phosphates.     

Selection of phytate-degrading human bifidobacteria and application in whole wheat dough fermentation

Lately, whole wheat products are highly recommended from their healthy properties. However, the presence of phytic acid (InsP(6)) could partly limit their benefits because it decreases the mineral bioavailability due to its chelating properties. The objective of this work was to select strains with high phytate-degrading activity from human feces, and evaluate their suitability for the bread making process. Twenty-three different bifidobacterial strains (13 from infants and 10 from adults) were isolated, belonging to the species Bifidobacterium longum, Bifidobacterium breve and Bifidobacterium catenulatum. The phosphatase and phytase activities of these strains were evaluated as well as their ability to degrade InsP(6) during growth. Then, the fermentative ability of the strain showing the highest phytate-degrading activity (B. longum. BIF307) was determined in whole wheat breadmaking. The use of the selected bifidobacterial strain as starter during whole wheat fermentation resulted in bread with similar technological quality than the control (in absence of bifidobacteria) and crumb with lower levels of inositol phosphates. Therefore, the used of the selected Bifidobacterium strain in whole wheat breadmaking process could provide potential nutritional benefits by decreasing the antinutrient content of the product.