Transgenic barley over-expressing Aspergillus niger phytase phyA in field trials

Effects of fungal phytase addition, formaldehyde treatment and dietary concentrate content on ruminal phosphorus availability

Background and ObjectivesGermination pretreatment is an effective way to improve the nutritional quality and sensory quality of germinated brown rice (GBR). Cold plasma pretreatment (CPP) has been demonstrated to improve the physicochemical properties of GBR. The effects of CPP on reducing phytic acid and improving nutrient composition in GBR have not been evaluated, and there are few studies on the changes of phytic acid and phytase, especially the changes of the forms and compositions of γ‐oryzanol, phenolics, and flavonoids in GBR with CPP. Therefore, this study evaluated the changes of phytase, phytic acid, γ‐aminobutyric acid (GABA), γ‐oryzanol, flavonoids, phenolics, and antioxidant activity in GBR with or without CPP.FindingsThe phytic acid content in CPP‐treated GBR for germination of 72 h was lower (7.60 mg/g, dry basis weight [DW]) than that in untreated GBR (9.01 mg/g DW). At the same germination time, the phytase activity and GABA, total γ‐oryzanol contents in CPP‐treated GBR were higher than those in untreated GBR. However, total flavonoids and phenolics levels, flavonoid compositions and phenolic acids contents, and T‐AOC and DPPH antioxidant capacity in CPP‐treated GBR were lower than those of untreated GBR.ConclusionsThese results indicated that CPP for brown rice was an effective method for decreasing the phytic acid and enhancing GABA and γ‐oryzanol in GBR compared with non‐CPP of brown rice for germination.Significance and noveltyCPP is beneficial to reduce phytic acid and improve the GABA and γ‐oryzanol contents of GBR, which provides a theoretical basis for producing functional and nutritious GBR foods.

ABSTRACTThe phytase (EC 3.1.3.8.) of Sanilac Navy Beans was extracted with 2% CaCl2 and purified by ammonium sulfate fractionation and DEAE‐cellulose chromatography. The enzyme showed an optimum pH of 5.3 and Km of 0.018 mM with phytic acid as substrate. The optimum temperature was 50° C. The activation energy of the enzymic hydrolysis of phytic acid was 11,500 Cal/mole and the inactivation energy of the enzyme 55,800 Cal/mole. The purified phytase showed broad specificity. This enzyme may be described as a nonspecific phosphomonoesterase with phytase and potent pyrophosphatase activities. It was inhibited by high concentrations of phytic acid. The activity was increased by about 35% in the presence of 1 mM Co++. Soaking of beans in distilled water did not affect their phytic acid content and phytase activity. Germination of the beans resulted in an increase in phytase activity and breakdown of phytic acid.

Biochemical characteristics of phytases from fungi and the transformed microorganism

Utilization of Phytic Acid by Cooperative Interaction in Rhizosphere

Phytate (inositol hexa phosphate) hydrolysis can occur during food preparation and production and in the intestine, either by phytase from plants, yeasts or other micro-organisms. This degradation is of nutritional importance, because removal of phosphate groups from the inositol ring results in an increased bioavailability of essential dietary minerals. To substantially improve iron absorption the degradation has to be virtually complete. Food processing techniques increasing the activity of the naturally occurring plant phytases are soaking, malting, hydrothermal treatment and fermentation. An alternative is addition of phytases or micro-organisms producing phytase. Phytate degradation in the stomach and small intestine occurs as a result of activity of dietary phytase of plant or microbial origin. The plant phytase is less stable than fungal phytase in the physiological conditions of the intestine. Biotechnologically produced phytases may be possible for use tomorrow in food processing as well as plant raw material and starter cultures with inserted phytase genes.

Seeds of twenty wheat cultivars grown with (+Zn = 23 kg Zn ha−1) and without zinc (Zn) fertilization in a Zn-deficient calcareous soil in Central Anatolia were analyzed for the levels of Zn, phosphorus (P), phytic acid, and phytase activity. Additionally, seeds of four wheat cultivars grown on 55 different locations in Turkey were also analyzed for Zn, P, and phytic acid. In the field experiment with 20 wheat cultivars, seed Zn concentrations showed a range between 7 to 11 mg kg−1 under Zn-deficient and 14 to 23 mg kg−1 under Zn-added conditions. Zinc fertilization reduced seed concentrations of P and phytic acid of all cultivars. On average, the reductions caused by Zn fertilization were from 3.9 to 3.5 mg g−1 for P and from 10.7 to 9.1 mg g−1 for phytic acid. Irrespective of Zn fertilization, seed phytic acid concentrations showed a large genotypic variation, i.e., from 7 to 12 mg g−1 with Zn fertilization and 8 to 13 mg g−1 at nil Zn treatment. As a result of decreases in phytic acid and increases in Zn concentrations by Zn fertilization, phytic acid to Zn molar ratios in seeds of cultivars markedly decreased. On average for all cultivars, phytic acid to Zn molar ratios decreased from 126 to 56 with Zn fertilization. Seed phytase activity of cultivars was not consistently influenced by varied Zn supply. However, on average for 20 cultivars, Zn fertilization tended to decrease phytase activity. In seeds of four wheat cultivars collected from 55 locations, the concentrations of Zn, P, and phytic acid ranged from 8 to 34 mg kg−1, 2.1 to 4.9 mg g−1, and 5.8 to 14.3 mg kg−1, respectively. Results obtained in the present study indicate that seed Zn concentrations of wheat cultivars grown in different locations of Turkey, especially under Zn-deficient conditions, are very low. Considering very high phytic acid : Zn molar ratios it can be suggested that bioavailability of Zn would be very low for humans. *Dedicated to the memory of the late Professor Dr. Ferhan Hatipoglu.

ABSTRACTWheat seedlings exhibited a differential ability to utilize P from a range of organic P substrates when grown in agar culture under sterile conditions. Plants showed limited ability to obtain P from inositol hexaphosphate (IHP), whereas other monoester substrates such as glucose 1‐phosphate (G1P), were equivalent sources of P for plant growth as compared with inorganic phosphate (Pi). Poor utilization of IHP was exemplified by significantly lower rates of dry matter accumulation and reduced P content of tissues, which were generally not significantly different to control plants that were grown in the absence of added P. The inability of wheat seedlings to obtain P from IHP was not associated with poor substrate availability but was due to either insufficient root phytase activity or inappropriate localization of phytase within root tissues. Phytase activities of 4 and 24 mU g−1 root fresh weight (FW) were determined for crude root extracts prepared from plants that were grown with either adequate P or under deficient conditions, respectively. Similar levels of phytase activity (approximately 12 mU g−1 FW) were observed in assays using intact roots, although no secreted activity was detected. By comparison, a secreted acid phosphomonoesterase activity was observed, and activities of between 466 and 1029 mU phosphomonoesterase g−1 root FW were measured for intact roots. On the basis of the differences in enzyme activity, and the observed differences in the ability of wheat seedlings to utilize G1P and IHP, it is evident that low intrinsic levels of phytase activity in wheat roots is a critical factor that limits the ability of wheat to obtain P from phytate when supplied in agar under non‐limiting conditions. This hypothesis was further supported by the observation that the ability of wheat to obtain P from IHP was significantly improved when the seedlings were inoculated with a soil bacterium (Pseudomonas sp. strain CCAR59) that possesses phytase activity.

SummaryThe phytase purple acid phosphatase (HvPAPhy_a) expressed during barley seed development was evaluated as transgene for overexpression in barley. The phytase was expressed constitutively driven by the cauliflower mosaic virus 35S‐promoter, and the phytase activity was measured in the mature grains, the green leaves and in the dry mature vegetative plant parts left after harvest of the grains. The T2‐generation of HvPAPhy_a transformed barley showed phytase activity increases up to 19‐fold (29 000 phytase units (FTU) per kg in mature grains). Moreover, also in green leaves and mature dry straw, phytase activities were increased significantly by 110‐fold (52 000 FTU/kg) and 57‐fold (51 000 FTU/kg), respectively. The HvPAPhy_a‐transformed barley plants with high phytase activities possess triple potential utilities for the improvement of phosphate bioavailability. First of all, the utilization of the mature grains as feed to increase the release of bio‐available phosphate and minerals bound to the phytate of the grains; secondly, the utilization of the powdered straw either directly or phytase extracted hereof as a supplement to high phytate feed or food; and finally, the use of the stubble to be ploughed into the soil for mobilizing phytate‐bound phosphate for plant growth.

Changes in the phytic acid, inorganic phosphorus and ATP contents, and in the activity of phytase and α‐amylase in rice (Oryza sativa L) grains were determined during 18 days of germination in a dark room. The effect of phytic acid on α‐amylase activity was studied in vitro. Rice grains immersed in sterilised deionised water at 14°C germinated on the fifth day. Phytase activity, detected in the ripening rice grains, increased linearly until the eighth day and reached a maximum on the tenth day. There was a marked decrease in phytate and an increase in inorganic phosphorus accompanying germination. There was a good inverse correlation between the levels of both phytase activity and inorganic phosphorus, and phytate breakdown. α‐Amylase activity was detected on the fourth day and increased markedly from the 12th to the 16th day of germination. ATP level increased from the second to the fourth day and slightly decreased from the fourth to the eighth day; it increased rapidly again from the eighth to the 18th day of germination. α‐Amylase activity was influenced by both pH and phytic acid concentration in the assay system. At 75 mM phytic acid, α‐amylase activity was lowered by 23%, 93% and 52% at pH 4–0, 5–0 and 6–0 respectively. When the enzyme, phytate and Ca2+ were incubated together at pH 5–0, the inhibition of α‐amylase by phytic acid was markedly decreased by addition of Ca2+. The chemical affinity of Ca2+ for phytic acid was higher in the reaction at pH 5–0 than in those at pH 4–0 and pH 6–0, and over 98% of Ca2+ in the reaction system was precipitated as Ca‐phytate.

To achieve high maize (Zea mays L.) yields and quality grain, it is necessary to develop stress-resistant cultivars and related cultivation practices, aiming to maximize efficiency. Thus, our objectives were (i) to investigate the impact of tillage practices and maize hybrids (which have improved over time) on yield and its components, and (ii) to characterize the response pattern of maize hybrid grain nutrient quality components to subsoiling. To achieve this, we conducted field trials with five maize hybrids from different eras under two tillage practices: rotary tillage and subsoiling. We compared grain yield, nutritional quality, and other indicators across different tillage conditions from the 1970s to the 2010s. The main results of this study are as follows: under rotary tillage conditions, the 2010s hybrid (DH618) significantly increased yields (9.37-55.89%) compared to hybrids from the 1970s-2000s. After subsoiling, the physiologically mature grains of all hybrids exhibited minimal changes in crude protein and fat content, while there was a significant reduction in the total soluble sugar content of the grains. After subsoiling, there was a substantial 8.14 to 12.79 percent increase in total starch accumulation in the grain for all hybrids during the period of 47-75 days post-anthesis. Furthermore, during the period of 47-75 days after anthesis, the consumption of grain crude protein significantly contributed to the accumulation of total starch in the grains. Ultimately, subsoiling significantly increased the yield of each hybrid and enhanced the total grain starch content at physiological maturity of all hybrids, with the 2010s hybrid (DH618) performing exceptionally well.

Distribution of phytic acid and phytase activity in pea seeds was analyzed and compared with those in wheat grains under identical assay conditions (55 °C, pH 5.5). Most total phytic acid content and phytase activity were found in pea cotyledons. In wheat grains, debranning resulted in a 70% reduction in phytic acid content, whereas more than 40% of the total phytase activity remained. The possibility to hydrolyze phytic acid by use of ground debranned wheat as a source of phytase in blends with pea cotyledon flour was investigated. The Michaelis-Menten parameters for each endogenous enzyme were identified and used to predict the rate of phytic acid hydrolysis. Results demonstrate a synergistic effect between the two phytase activities, enabling a 70-95% reduction of phytic acid depending on pea/wheat flour ratios in a relatively short time (4 h). This reduction appears to be able to increase zinc bioavailability but remains insufficient for iron.

The Qinghai-Tibetan Plateau in China is considered to be one of the original centers of cultivated barley. At present, little is known about the phytase activity (Phy) or phytic acid content (PA) in grains of Tibetan annual wild barley. Phy and PA were determined in grains of 135 wild and 72 cultivated barleys. Phy ranged from 171.3 to 1299.2 U kg(-1) and from 219.9 to 998.2 U kg(-1) for wild and cultivated barleys, respectively. PA and protein contents were much higher in wild barley than in cultivated barley. Tibetan annual wild barley showed a larger genetic diversity in phytase activity and phytic acid and protein contents and is of value for barley breeding. There is no significant correlation between phytase activity and phytic acid or protein content in barley grains, indicating that endogenous phytase activity had little effect on the accumulation of phytic acid.

Enhanced and suppressed phosphorus mineralization by Ca complexation: NMR and CD spectroscopy investigation

Phytate is the major storage form of phosphorus in seeds and so is a common dietary constituent. Excessive ingestion of undegraded phytates can cause mineral deficiencies in humans. In addition, phytic acid is antineoplastic in animal models of both colon and breast carcinoma. There have been no previous studies quantifying phytase activity in the human small intestine although it is present in animals. Small intestinal phytase and alkaline phosphatase activity and distribution was measured in vitro in mucosal homogenates from two human small intestinal specimens obtained from transplant donors. Rat intestine was also studied for comparison. Phytase activity was found in human small intestine at low values (30 times less than that in rat tissue and 1000-fold lower than alkaline phosphatase in the same tissue). The activity was greatest in the duodenum and lowest in the ileum. In conclusion, the normal human small intestine has very limited ability to digest undegraded phytates. Although this may have adverse nutritional consequences with respect to metabolic cation imbalances, the presence of undigested phytate in the colon may protect against the development of colonic carcinoma.