Phytases of Probiotic Bacteria: Characteristics and Beneficial Aspects

Probiotics play a vital role in clinical applications for the treatment of diarrhea, obesity and urinary tract infections. Phytate, an anti-nutrient, chelates essential minerals that are vital for human health. In the past few decades, research reports emphasize extensively on phytate degradation in animals. There is a growing need for finding alternate strategies of phytate utilization in human, as they are unable to produce phytase. At this juncture, probiotics can be utilized for phytase production to combat mineral deficiency in humans. The main focus of this review is on improving phosphate bioavailability by employing two approaches: supplementation of (1) fermented food products that contain probiotics and (2) recombinant phytase producing bacteria. In addition, several factors influencing phytase activity such as bacterial viability, optimal pH, substrate concentration and specificity were also discussed.     

Dietary molar ratios of phytate: Zinc and millimolar ratios of phytate × calcium: Zinc in South Koreans

The zinc nutritional status in south Koreans was established by evaluation of zinc, calcium and phytate intakes, the molar ratio of phytate: zinc, and the millimolar ratio of phytate × calcium: zinc. The intakes of iron and magnesium were also estimated. Sampling was designed so that it was representative of the national population. Two-day food records were used for the calculation of nutrient intakes, using food consumption data from the 1995 National Nutrition Survey (’95NNS) for South Korea. Daily intakes of zinc and calcium were estimated to be 10.1 mg/d and 426.5 mg/d, respectively, and those of iron and magnesium were 15.2 mg/d and 268.0 mg/d, respectively. The estimated daily phytate intake was 1676.6 mg/d. The ratio of phytate: zinc was 15.9 mol/d and that of phytate × calcium: zinc was 168.9 mmol/d. The ratio in millimoles per 4.2 MJ (1000 kcal) of phytate × calcium: zinc was 91.8. The major food groups for zinc intake were meat, poultry and their products (43%), and cereals and grain products (18%). Sixty-two percent of zinc was from animal food sources. Cereal and grain products supplied most of the phytate intake (46%) followed by seasonings, fruits, and legumes and their products. The major food source of phytate was rice (39%). The results of the study raise concern about the suboptimal zinc status in relation to the Western diet.     

Bioavailability of iron, zinc, phytate and phytase activity during soaking and germination of white sorghum varieties

The changes in phytate, phytase activity and in vitro bioavailability of iron and zinc during soaking and germination of three white sorghum varieties (Sorghum bicolor L. Moench), named Dorado, Shandweel-6, and Giza-15 were investigated. Sorghum varieties were soaked for 20 h and germinated for 72 h after soaking for 20 h to reduce phytate content and increase iron and zinc in vitro bioavailability. The results revealed that iron and zinc content was significantly reduced from 28.16 to 32.16% and 13.78 to 26.69% for soaking treatment and 38.43 to 39.18% and 21.80 to 31.27% for germination treatments, respectively. Phytate content was significantly reduced from 23.59 to 32.40% for soaking treatment and 24.92 to 35.27% for germination treatments, respectively. Phytase enzymes will be activated during drying in equal form in all varieties. The results proved that the main distinct point is the change of phytase activity as well as specific activity during different treatment which showed no significant differences between the varieties used. The in vitro bioavailability of iron and zinc were significantly improved as a result of soaking and germination treatments.     

Barley HvPAPhy_a as transgene provides high and stable phytase activities in mature barley straw and in grains

The 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.     

Biotechnological production and applications of phytases

Phytases decompose phytate, which is the primary storage form of phosphate in plants. More than 10 years ago, the first commercial phytase product became available on the market. It offered to help farmers reduce phosphorus excretion of monogastric animals by replacing inorganic phosphates by microbial phytase in the animal diet. Phytase application can reduce phosphorus excretion by up to 50%, a feat that would contribute significantly toward environmental protection. Furthermore, phytase supplementation leads to improved availability of minerals and trace elements. In addition to its major application in animal nutrition, phytase is also used for processing of human food. Research in this field focuses on better mineral absorption and technical improvement of food processing. All commercial phytase preparations contain microbial enzymes produced by fermentation. A wide variety of phytases were discovered and characterized in the last 10 years. Initial steps to produce phytase in transgenic plants were also undertaken. A crucial role for its commercial success relates to the formulation of the enzyme solution delivered from fermentation. For liquid enzyme products, a long shelf life is achieved by the addition of stabilizing agents. More comfortable for many customers is the use of dry enzyme preparations. Different formulation technologies are used to produce enzyme powders that retain enzyme activity, are stable in application, resistant against high temperatures, dust-free, and easy to handle.     

Effect of calcium,copper, and zinc levels in a rapeseed meal diet on mineral and trace element utilization in the rat

Mineral and trace element interactions were studied in a balance trial with rats. Calcium, copper, and zinc were supplied to a rapeseed meal diet in a factorial design. Animals were fedad libitum, and absorption, excretion, and retention of the elements were evaluated either as fractions of total intake or in relation to nitrogen retention to account for differences in food intake and lean body mass increment. The intrinsic content of minerals and trace elements was sufficient to support growth at a rate that could be expected from the rapeseed protein quality. However, when calcium was included in the diet, the intrinsic dietary level of zinc appeared to be limiting, despite the fact that the zinc level was twice the recommended level. Additional zinc supply reversed growth impairment. This calcium-zinc interaction is believed to be owing to the formation of phytate complexes. Calcium addition influenced the calcium, phosphorus, magnesium, zinc, and iron—but not the copper—balances. The addition of calcium reduced the availability of the intrinsic zinc, whereas no effect was seen in the zinc-fortified groups. The availability of intrinsic copper was in a similar way significantly impaired by addition of dietary zinc, where-as copper-supplied groups were unaffected by zinc addition. Intrinsic iron availability was also dependent upon zinc addition, although in a more ambigouus way. Thus, addition of extrinsic minerals to a diet high in phytate can result in significant impairments of growth and mineral utilization.     

Dephytinization of a rat diet

Soaking of a rat diet, high in both plant phytate and phytase, progressively degraded the phytate content with time of soaking. This dephytinization in turn enhanced the digestion of feed organic matter in the animals, and it significantly improved the absorption and retention of minerals and trace elements as observed in balance studies. Incorporation of elements into specific tissues was evaluated as a reflection of bioavailability. Some tissues did reflect the preceding absorption of certain elements; other tissues seemed less suitable as indicators of trace element absorption. Dietary calcium addition in many ways contrasted the soaking procedure: inorganic calcium addition to the feed reduced phosphorus, magnesium, and trace element bioavailability, and interfered with the internal deposition of the elements. The external dephytinization of the feed did not affect the phosphohydrolase activity of the intestinal mucosa as manifested by alkaline phosphatase activity or phytase activity. The mucosal phytase and alkaline phosphatase activities were, however, mutually correlated, supporting the view that “phytase” activity is a less substratespecific action of alkaline phosphatase activity or a fraction of this activity.     

Screening of phytase producers and optimization of culture conditions for submerged fermentation

Phytase (myo-inositol-hexakisphosphate phosphohydrolase) is an enzyme, which breaks down phytate to inositol and orthophosphoric acid. Phytase has been used as feed additive, and in some medical applications for years. To date, phytase production has been usually performed as a solid-state fermentation with small production volumes. Therefore, the aim of this study was to increase the phytase activity in submerged fermentations by screening several microorganism strains based on the literature to select the most productive phytase producer and optimizing growth parameters such as temperature, pH, and aeration level using response surface methodology (RSM). As a result, among the four different microorganisms evaluated, Aspergillus ficuum (NRRL 3135) was selected as the most productive strain. Optimum temperature, pH, and aeration values were determined as 33 °C, 4.5, and 0.9 vvm, respectively, for A. ficuum in 2-l batch submerged phytase productions. Under these conditions, phytase activity was measured as 2.27 U/ml. Therefore, this is a unique study showing the production of phytase with A. ficuum successfully in submerged fermentation as opposed to the traditional solid-state fermentation.     

The phytate and mineral content of some cereals,cereal products,legumes, legume products,snack bars,and nuts available in New Zealand

Analyses for phytate by an indirect precipitation method and for the minerals calcium (Ca), zinc (Zn), iron (Fe), copper (Cu), and manganese (Mn) by atomic absorption spectrophotometry were carried out on 100 foods available in New Zealand. Foods with 1% phytate (dry weight basis) included untoasted muesli, rolled oats, wheat germ, wheat bran, soybean, and some soy products. Most breads contained between 0.35 and 0.60% phytate; legumes on average had 0.62% phytate, as did snack bars. There was a wide variation in Ca and Zn contents: There was a tenfold variation in Ca content among the legume products, whereas there was a seventyfold variation in Zn content among the cereals. The phytate: Zn molar ratio, which is presumed to indicate the biovailability of Zn, was above 20∶1 for two-thirds of the cereals and almost all of the snack bars; it was above 15∶1 for one-third of the breads, almost all of the legumes, and half of the legume products. These high phytate: Zn molar ratios, as well as some Ca: phytate molar ratios above 6∶1, indicate that there might be a reduced biovailability of Zn in many of the foods analyzed in this study.     

Dietary Fiber Intake Increases the Risk of Zinc Deficiency in Healthy and Diabetic Women

Phytic acid is a major determinant of zinc bioavailability. Little is known about phytic acid intakes or indices of zinc bioavailability in type 2 diabetes mellitus (DM), a condition that predisposes to zinc deficiency. The aim of this cross-sectional study was to measure and explore the relationships among phytic acid intake, zinc bioavailability, and molecular markers of zinc homeostasis in 20 women with DM compared to 20 healthy women. The phytate/zinc, (calcium)(phytate)/zinc, and (calcium + magnesium)(phytate)/zinc molar ratios were used to indicate zinc bioavailability. Plasma zinc concentrations and zinc transporter (ZnT1, ZnT8, and Zip1) gene expression in mononuclear cells were measured. Participants with DM consumed 1,194?±?824?mg/day (mean?±?SD) phytic acid, an amount similar to the intake of healthy women (1,316?±?708?mg/day). Bread products and breakfast cereals contributed more than 40?% of the phytic acid intake in each group. A positive relationship was observed in all participants between phytic acid and dietary fiber (r?=?0.6, P?<?0.001) and between dietary fiber and the (calcium)(phytate)/zinc ratio (r?=?0.5, P?<?0.001). Compared to the healthy group, the messenger RNA ratio of ZnT1 (zinc export) to Zip1 (zinc import) was lower in participants with DM, which may indicate perturbed zinc homeostasis in the disorder. The plasma zinc concentration was not predicted by age, body mass index, health status, zinc bioavailability, or zinc transporter expression. Healthy and diabetic women consume phytic acid in amounts that are likely to decrease the bioavailability of dietary zinc. Recommendations to consume greater amounts of dietary fiber, much of which is associated with phytate, increase the risk of zinc deficiency.     

Use of Lactobacilli in Cereal-Legume Fermentation and as Potential Probiotics towards Phytate Hydrolysis

Phytate is a potent inhibitor of mineral absorption in humans occurring in plant-based food. Application of lactobacilli that produce phytate-degrading enzymes (phytases) to reduce phytate is an interesting yet a not much explored sector of research. Therefore, phytate dephosphorylation by Lactobacillus plantarum MTCC 1325 was evaluated. Cells at stationary phase showed phytase activity which was maximal at 24 h of growth. Glucose concentration and the type of phosphorous source in the media modulated the enzyme activity. Fermentation of cereal and/or legume flours with the strain resulted in phytate reduction with the highest in sorghum (73%) and the lowest in horse gram (34%). Further, the strain showed tolerance to acid, bile, and simulated gastrointestinal fluid. Significant phytase activity in the presence of simulated gastrointestinal fluids along with the ability to produce phytases post-exposure to simulated gastrointestinal fluids is of interest. To the best of our knowledge, this is the first report on the effect of simulated gastrointestinal fluid on cell-associated phytases of lactobacilli. The results of the investigation indicate that L. plantarum MTCC 1325 could be used as a starter in cereal-legume fermentation and as potential probiotics to achieve phytate hydrolysis in food matrices and also in gastrointestinal tract.     

In Vitro Bioavailability of Calcium,Magnesium, Iron,Zinc, and Copper from Gluten-Free Breads Supplemented with Natural Additives

The aim of this study was to measure the content of calcium, magnesium, iron, zinc, and copper and determine the bioavailability of these ingredients in gluten-free breads fortified with milk and selected seeds. Due to the increasing prevalence of celiac disease and mineral deficiencies, it has become necessary to produce food with higher nutritional values which maintains the appropriate product characteristics. This study was designed for gluten-free breads fortified with milk and seeds such as flax, poppy, sunflower seeds, pumpkin seeds or nuts, and flour with amaranth. Subsequently, digestion was performed in vitro and the potential bioavailability of the minerals was measured. In the case of calcium, magnesium, iron, and copper, higher bioavailability was observed in rice bread, and, in the case of copper and zinc, in buckwheat bread. This demonstrated a clear increase in bioavailability of all the minerals when the bread were enriched. However, satisfactory results are obtained only for the individual micronutrients.     

Purification and Biochemical Characterization of Phytase Enzyme from <Emphasis Type="Italic">Lactobacillus coryniformis</Emphasis> (<Emphasis Type="Italic">MH121153</Emphasis>)

Phytase (myo-inositol hexaphosphate phosphohydrolase) belongs to phosphatases. It catalyzes the hydrolysis of phytate to less-phosphorylated inorganic phosphates and phytate. Phytase is used primarily for the feeding of simple hermit animals in order to increase the usability of amino acids, minerals, phosphorus and energy. In the present study, phytase isolation from the Lactobacillus coryniformis strain, isolated from Lor cheese sources, phytase purification and characterization were studied. The phytase was purified in simple three steps. The enzyme was obtained with 2.60% recovery and a specific activity of 202.25 (EU/mg protein). The molecular mass of the enzyme was determined to be 43.25 kDa with the sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) method. The optimum temperature and pH for the enzyme were found as 60 °C and 5.0 and respectively. To defined the substrate specificity of the phytase, the hydrolysis of several phosphorylated compounds by the purified enzyme was studied and sodium phytate showed high specificity. Furthermore, the effects of Ca²⁺, Ag⁺, Mg²⁺, Cu²⁺, Co²⁺, Pb²⁺, Zn²⁺ and Ni²⁺ metal ions on the enzyme were studied.     

Expression of an Aspergillus niger Phytase Gene (phyA) in Saccharomyces cerevisiae

Phytase improves the bioavailability of phytate phosphorus in plant foods to humans and animals and reduces phosphorus pollution of animal waste. Our objectives were to express an Aspergillus niger phytase gene (phyA) in Saccharomyces cerevisiae and to determine the effects of glycosylation on the phytase’s activity and thermostability. A 1.4-kb DNA fragment containing the coding region of the phyA gene was inserted into the expression vector pYES2 and was expressed in S. cerevisiae as an active, extracellular phytase. The yield of total extracellular phytase activity was affected by the signal peptide and the medium composition. The expressed phytase had two pH optima (2 to 2.5 and 5 to 5.5) and a temperature optimum between 55 and 60°C, and it cross-reacted with a rabbit polyclonal antibody against the wild-type enzyme. Due to the heavy glycosylation, the expressed phytase had a molecular size of approximately 120 kDa and appeared to be more thermostable than the commercial enzyme. Deglycosylation of the phytase resulted in losses of 9% of its activity and 40% of its thermostability. The recombinant phytase was effective in hydrolyzing phytate phosphorus from corn or soybean meal in vitro. In conclusion, the phyA gene was expressed as an active, extracellular phytase in S. cerevisiae, and its thermostability was affected by glycosylation.     

Novel Glucose-1-Phosphatase with High Phytase Activity and Unusual Metal Ion Activation from Soil Bacterium Pantoea sp. Strain 3.5.1

Phosphorus is an important macronutrient, but its availability in soil is limited. Many soil microorganisms improve the bioavailability of phosphate by releasing it from various organic compounds, including phytate. To investigate the diversity of phytate-hydrolyzing bacteria in soil, we sampled soils of various ecological habitats, including forest, private homesteads, large agricultural complexes, and urban landscapes. Bacterial isolate Pantoea sp. strain 3.5.1 with the highest level of phytase activity was isolated from forest soil and investigated further. The Pantoea sp. 3.5.1 agpP gene encoding a novel glucose-1-phosphatase with high phytase activity was identified, and the corresponding protein was purified to apparent homogeneity, sequenced by mass spectroscopy, and biochemically characterized. The AgpP enzyme exhibits maximum activity and stability at pH 4.5 and at 37°C. The enzyme belongs to a group of histidine acid phosphatases and has the lowest K_m values toward phytate, glucose-6-phosphate, and glucose-1-phosphate. Unexpectedly, stimulation of enzymatic activity by several divalent metal ions was observed for the AgpP enzyme. High-performance liquid chromatography (HPLC) and high-performance ion chromatography (HPIC) analyses of phytate hydrolysis products identify dl-myo-inositol 1,2,4,5,6-pentakisphosphate as the final product of the reaction, indicating that the Pantoea sp. AgpP glucose-1-phosphatase can be classified as a 3-phytase. The identification of the Pantoea sp. AgpP phytase and its unusual regulation by metal ions highlight the remarkable diversity of phosphorus metabolism regulation in soil bacteria. Furthermore, our data indicate that natural forest soils harbor rich reservoirs of novel phytate-hydrolyzing enzymes with unique biochemical features.     

Quantitative conversion of phytate to inorganic phosphorus in soybean seeds expressing a bacterial phytase

Phytic acid (PA) contains the major portion of the phosphorus in the soybean (Glycine max) seed and chelates divalent cations. During germination, both minerals and phosphate are released upon phytase-catalyzed degradation of PA. We generated a soybean line (CAPPA) in which an Escherichia coli periplasmic phytase, the product of the appA gene, was expressed in the cytoplasm of developing cotyledons. CAPPA exhibited high levels of phytase expression, >or=90% reduction in seed PA, and concomitant increases in total free phosphate. These traits were stable, and, although resulted in a trend for reduced emergence and a statistically significant reduction in germination rates, had no effect on the number of seeds per plant or seed weight. Because phytate is not digested by monogastric animals, untreated soymeal does not provide monogastrics with sufficient phosphorus and minerals, and PA in the waste stream leads to phosphorus runoff. The expression of a cytoplasmic phytase in the CAPPA line therefore improves phosphorus availability and surpasses gains achieved by other reported transgenic and mutational strategies by combining in seeds both high phytase expression and significant increases in available phosphorus. Thus, in addition to its value as a high-phosphate meal source, soymeal from CAPPA could be used to convert PA of admixed meals, such as cornmeal, directly to utilizable inorganic phosphorus.     

Bioavailability of zinc sources and their interaction with phytates in broilers and piglets

Zinc (Zn) is essential for swine and poultry and native Zn concentrations in feedstuffs are too low to meet their Zn requirement. Dietary Zn bioavailability is affected by phytate, phytase and Zn supplemented in organic form is considered as more bioavailable than inorganic sources. A meta-analysis using GLM procedures was processed using broiler and piglet databases to investigate, within the physiological response of Zn, (1) the bioavailability of inorganic and organic Zn sources (Analysis I); (2) the bioavailability of native and inorganic Zn dependent from dietary phytates, vegetal and supplemental phytase activity (Analysis II). Analysis I: the bioavailability of organic Zn relative to inorganic Zn sources ranged, depending on the variable, from 85 to 117 never different from 100 (P > 0.05). The coefficients of determination of the regressions were 0.91 in broilers and above 0.89 in piglets. Analysis II: in broilers, bone Zn was explained by supplemental Zn (linear and quadratic, P < 0.001) and by supplemental phytase (linear, P < 0.001). In piglets, the interaction between dietary Zn and phytates/phytases was investigated by means of a new variable combining dietary phytic phosphorus (PP) and phytase activity. This new variable represents the remaining dietary PP after its hydrolysis in the digestive tract, mainly due to phytase and is called non-hydrolyzed phytic phosphorus (PP_NH). Bone Zn was increased with native Zn (P < 0.001), but to a lower extent in high PP or low phytase diets (ZN_N × PP_NH, P < 0.001). In contrast, the increase in bone zinc in response to supplemental Zn (P < 0.001) was not modulated by PP_NH (P > 0.05). The coefficients of determination of the regressions were 0.92 in broilers and above 0.92 in piglets. The results from the two meta-analyses suggest that (1) broilers and piglets use supplemented Zn, independent from Zn source; (2) broiler use native Zn and the use is slightly enhanced with supplemental phytase; (3) however, piglets are limited in the use of native Zn because of the antagonism of non-hydrolyzed dietary phytate. This explains the higher efficacy of phytase in improving Zn availability in this specie.     

Phytate degradation by micro-organisms in synthetic media and pea flour

AIMS: To screen micro-organisms for the ability to produce phytase enzyme(s) and to use promising strains for the fermentation of pea flour. METHODS AND RESULTS: Two methods using the indirect estimation of phytate degradation were evaluated and both shown to be inadequate. A third method, measuring the inositol phosphate (IP3-IP6) content directly during fermentation, was used instead of the indirect estimations of phytate degradation. In synthetic media, some strains required customized conditions, with no accessible phosphorus sources other than phytate, to express phytase activity. The repression of phytase synthesis by inorganic phosphorus was not detected during fermentation with pea flour as substrate and seemed to be less significant with a higher composition complexity of the substrate. None of the tested lactic acid bacteria strains showed phytase activity. CONCLUSIONS: The methodology for the phytase screening procedure was shown to be critical. Some of the screening methods and media used in previous publications were found to be inadequate. SIGNIFICANCE AND IMPACT OF THE STUDY: This paper highlights the pitfalls and difficulties in the evaluation of phytase production by micro-organisms. The study is of great importance for future studies in this area.     

Genome-wide association analysis of nutritional composition-related traits and iron bioavailability in cooked dry beans (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L.)

Seed nutrients in legumes are important for human health, particularly in developing countries with heavy reliance on plant-based diets, and among vegetarians in developed nations. Here, we report on our efforts to uncover the genetic basis underlying the phenotypic variation for protein, zinc, calcium concentrations, and iron bioavailability present in 206 accessions of dry bean (Phaseolus vulgaris L.) from the Andean Diversity Panel (ADP). We used 8111 single nucleotide polymorphisms (SNPs) generated with genotyping-by-sequencing (GBS) to examine the allelic variants’ associations with seed protein, zinc, and calcium concentrations, and iron bioavailability in the 206 ADP accessions grown over 2 years in Michigan. These efforts identified phenotypic variation among the ADP genotypes for each of the traits, with the highest variation (5.4-fold) found for cooked seed iron bioavailability. In addition, significant SNP-trait associations were found and explained from 6.3 to 13.2% of the phenotypic variation. These results expand the current understanding of the genetic architecture underlying these complex nutritional quality traits and iron bioavailability in dry beans. Furthermore, they have utility for future nutritional quality breeding efforts to better biofortify dry bean through genomics-assisted breeding.