mammmnmnmnm i ,W. ~ -- 93 °°85° 3504 : mmr This is to certify that the dissertation entitled YEAST PHYTASE AND WHEAT INOSITOL PHOSPHATES presented by NARSIMHA REDDY NAY l NI has been accepted towards fulfillment of the requirements for PH.D. degreein FOOD SCLENCE «2: HUMAN NUTRITION 21:1. {44-2/4 (7 2126.41” Major professor Date 11-11-83 MSU is an Affirmative Action/Equal Opportunity Institution 0-12771 PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before date due. DATE DUE DATE DUE DATE DUE JUN i 379:9: —————Jl L________ l _l MSU Is An Affirmative Action/Equal Opportunity Institution encircmuna-pd YEAST PHYTASE AND WHEAT INOSITOL PHOSPHATES BY Narsimha Reddy Nayini A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Food Science and Human Nutrition 1983 ABSTRACT YEAST PHYTASE AND WHEAT INOSITOL PHOSPHATES By Narsimha Reddy Nayini Wheat contains phytic acid, myoinositol 1, 2, 3, 4, 5, 6-hexakis (di-hydrogen phosphate), which may bind minerals and reduce their bioavailability. Enzymes present in bread yeast and plant tissues can hydrolyze phytic acid to phos- phoric acid and inositol through a series of intermediate inositol phosphates. The objectives of this research were to study (a) the properties of yeast phytase (b) the effect of milling ex- traction rate and bread fermentation time on the entire spectrum of inositol phosphates present in wheat flour and bread and (c) the metal-binding properties of the wheat inositol phosphates. The yeast phytase was extracted with 2% CaC12, purified by ammonium sulfate fractionation and DEAE-cellulose chro- matography. The phytase showed an optimum pH of 4.6 and optimum temperature of 45°C with phytic acid as substrate. 2+ The enzyme activity is increased by 1 mM of Fe and de- creased by chelating agents. The yeast phytase had Km = 0.21 mM with phytate as substrate. The enzyme shows broad specificity and hydrolyzes several phosphomonoesters besides phytic acid and other inositol phosphates. It is a non- specific phosphomonoesterase characterized by potent pyro- phosphatase activity. Hexa- (1P6), penta— (1P5), tetra- (1P4), tri- (1P3), di- (1P2), mono-phosphate (1P1) along with inoréganic phosphate (P1) were found in all flours and breads studied. Inositol hexaphosphate, inorganic phosphate and total phos- phorus increased as the extraction rate increased from 70% to 90% to 100%. When the doughs were subjected to various fermentation times, from 0 to 120 min, a decrease in IP6 and an increase in P1 were observed. There were always, however, intermediate inositol phosphates which did not follow any trend in their quantitative changes, but an overall phosphate balance could be obtained only by con- sidering their presence. Commercial white bread contained more Pi’ IPG and total phosphorus than expected, assuming a 70-75%-extraction, because of added phosphates which probably slowed down the hydrolysis of 1P6. The distribution of inositol phosphates in commercial whole wheat bread was 17.4% IP 20.0% IP 6’ and 25.6% Pi. 5’ 13.0% 1P4, 12.0% IP 6.5% IP 5.5% IP 3’ 2) 1 All six wheat inositol phosphates and phosphoric acid showed the ability to precipitate the minerals Ca, Cu, Fe, and Zn at pH's 4, 5 and 6, except IP1 and phosphoric acid which did not precipitate zinc at pH 4. With a few excep- tions, the phosphorus to calcium atom ratio was 1 : 1 in the inositol phosphates isolated from wheat. Iron and copper showed a decrease in the ratio of phosphorus to metal as the pH rose from 4 to 5. With a rise in pH, zinc showed an increase in the P : Zn ratio in IP2 and 1P3, no change in IP5 and a decrease in IP4 and 1P6. ACKNOWLEDGMENTS The author is deeply indebted to his major professor Pericles Markakis for his assistance in conducting this study, encouragement and patience throughout the course of this work. Appreciation is also expressed to professors Robert J. Brunner, Jerry N. Cash, Wanda L. Chenoweth from the department of Food Science and Human Nutrition and David R. Dilley of the department of Horticulture for their critical evaluation of this manuscript. The author is most greatful to his brother Dr. Krishna R. Nayini and sister in-law Dr. Rama R.'Nayini for their encouragement throughout the graduate program. ii TABLE OF CONTENTS Page LIST OF TABLES . . . . . . . . . . . . . . . . . . . . v LIST OF FIGURES . . . . . . . . . . . . . . . . . . . . vii INTRODUCTION . . . . . . . . . . . . . . . . . . . . . 1 LITERATURE RIVIEW . . . . . . . . . . . . . . . . . . . 3 Phytase enzyme . . . . . . . . . . . . . . . . 3 Chemistry of phytic acid . . . . . . . . . . . 7 Biochemistry of phytic acid . . . . . . . . . . . 8 Occurance . . . . . . . . . . . . . . . . . . 9 Physiological role . . . . . . . . . . . . . . . 12 Protein- -phytate complex . . . . . . . . . . 15 Destruction of phytic acid during breadmaking . 17 Nutritional aspects of inositol phosphates . 19 MATERIALS AND METHODS . . . . . . . . . . . . . . . . . 24 Enzyme extraction . . . . . . . . . . . . . . . 24 Ammonium sulfate fractionation . . . . . . . . . 24 Assay procedure . . . . . . . . . . . 25 Determination of inorganic phosphorus by the ascorbic acid method . . . . . . . . . . . . . 26 Effect of pH on enzyme activity . . . . . . . 27 Effect of temperature on enzyme activity . . . . 27 Effect of substrate concentration on reaction rate . . . . . . . . . . . . . . . . . 27 Effect of various metal ions on phytase activity . . . . . . . . . . . 28 Effect of chelating agents on phytase activity . . . . . . . . . . . . 28 Separation of phytase from phosphatase . . . . . 29 Substrate specificity . . . . . . . . . . . . . . 30 Preparation of bread . . . . . . . . . . . . . . 30 Separation of inositol phosphates . . . . . .. 32 Phosphorus determination of chromatographic fractions . . . . . . . . . 32 Inositol determination of chromatographic fractions . . . . . . . . . . . . . . . . . . . 35 iii Page Inositol phosphate collection . . . . . . . . . 36 Precipitation of inositol phosphate-metal complex . . . . . . . . . . . . . . . . . . . 36 Analysis of Calcium . . . . . . . . . . . . . . 37 Analysis of Copper . . . . . . . . . . . . . . . 38 Analysis of Iron . . . . . . . . . . . . . . . 39 Analysis of Zinc . . . . . . . . . . . . . . . 40 RESULTS AND DISCUSSION . . . . . . . . . . . . . . . 42 Yeast phytase extraction . . . . . . . . . . . . 42 Effect of pH and temperature . . . . . 42 Effect of substrate concentrations on activity . 45 Effect of metal ions on yeast phytase activity . 47 Effect of chelating agents on yeast phytase activity . . . . . . . . . . . 50 Separation of phytase from phosphatase . . . . . 51 Substrate specificity of yeast phytase . . . . . 51 Effect of extraction rates on inositol phosphates in wheat flours and breads . . . . 54 Effect of fermentation time on inositol phosphates in wheat flours and breads . . . . 58 Inositol phosphates in commercial breads . . . . 65 Inositol phosphates-metal binding. . . . . . . . 65 Inositol monophosphate-metal binding . . . . . . 65 Inositol diphosphate-metal binding . . . . . . . 68 Inositol triphosphate-metal binding . . . . . . 70 Inositol tetraphosphate-metal binding . . . . . 72 Inositol pentaphosphate-metal binding . . . . . 74 Inositol hexaphosphate-metal binding . . . . . . 74 Phosphoric acid-metal binding . . . . . . . . . 77 Possible structures of inositol phosphate-metal complex . . . . . . . . . . . . . . . . . . . 80 CONCLUSIONS . . . . . . . . . . . . . . . . . . . . . 84 BIBLIOGRAPHY . . . . . . . . . . . . . . . . . . . . . 89 APPENDIX . . . . . . . . . . . . . . . . . . . . . . . 97 iv Table 10. 11. LIST OF TABLES Effect of metal ions on the yeast phytase activity . . . . . . . . . . . . . . . Effect of chelating agents on the yeast phytase activity . . . . . . . . . . Substrate specificity of purified yeast phytase . . . . . . . . . . . . . . . Ratio of phosphorus inositol in fractions obtained from Dowex 1x8 (Cl‘) chromatography . . . . . . . . . . . . . Concentration inositol phosphates and inorganic phosphates in flours of varying extraction rates and breads made from them . . . . . . . . Wheat flour bread contents in inositol phosphates as a function of fermentation time 0 0 O O O O 0 O I O O O O O O O 0 Concentration of inositol phosphates and inorganic phosphate in the commercial white and whole wheat bread . Percentages of metal and phosphorus precipitated from inositol monophosphate solution at pH's 4, 5 and 6 . . . Percentages of metal and phosphorus precipitated from inositol diphosphate solution at pH's 4, 5 and 6 . . . . Percentages of metal and phosphorus precipitated from inositol triphosphate solution at pH's 4, 5 and 6 . . Percentages of metal and phosphorus precipitated from inositol tetraphosphate solution at pH's 4, 5 and 6 . Page 49 50 53 54 57 60 66 67 69 71 73 Table Page 12. Percentages of metal and phosphorus precipitated from inositol pentaphosphate solution at pH's 4, 5 and 6 . . . . 75 13. Percentages of metal and phosphorus precipitated from inositol hexaphosphate solution at pH's 4, 5 and 6 . . . . . . . . 76 14. Percentages of metal and phosphorus precipitated from phosphoric acid solution at pH's 4, 5 and 6 . . . . . . . . . . . . . 78 vi Figure 10. 11. 12. LIST OF FIGURES Anderson's structure of phytic acid . . . Longitudinal section of wheat kernel Apparatus used for evaporating liquid in tubes . . . . . . . . Effect of pH on the yeast phytase activity Effect of temperature on yeast phytase. activity . . . . . . . . . . . . Effect of substrate concentrations on yeast phytase activity . . . . Lineweaver-Burk plot for yeast phytase with sodium phytate as substrate DEAE-cellulose chromatography of yeast extract . . . . . . . . . . . . . Elution pattern of inositol phosphates on Dowex 1x8 (Cl') column Scheme of dephosphorylation of phytic acid by phytase (Tomlinson and Ballou) Effect of fermentation time on the phytic acid and Pi content of whole wheat bread Effect of fermentation time on the phytic acid and Pi content of wheat bread vii Page 13 33 43 44 46 48 52 56 61 63 64 INTRODUCTION Phytic acid has become the subject of active research for sometime because of its influence in both functional and nutritional properties of foods. A great number of studies have been conducted to understand its biochemistry and physiological role in plants and animals. Phytates, which represent a complex class of naturally occurring compounds, are the main source of phosphorus in many seeds. Extensive use of cereals and legumes is a definite- hope for reducing the hunger and malnutrition of the world. As the world pupulation increases so increases the demand for protein. Plant proteins, however, are frequently asso- ciated wiht metabolic inhibitors and phytic acid is one of them. Phytic acid affects solubility and other character-' estics of protein. Phytic acid also chelates important minerals (Zn, Fe, Ca, Cu) and reduces their bioavailability. Wheat, as all cereal grains, contain phytic acid. During the bread fermentation, enzymes present in the yeast hydrolyse phytic acid to inositol phosphates poorer in phosphorus than phytic acid and finally to inositol and phosphoric acid. Although extensive work has been done on phytic acid and its metal-binding properties, little is known about the properties of the yeast phytase, distribution of intermediate inositol phosphates in flour and bread and their chelating characteristics. The objectives of this research were to study (a) the purification and properties of yeast phytase (b) the effect of extraction rate on the wheat flour (c) the effect of fermentation time on the inositol phosphates present in bread and (d) the mineral-binding properties of the wheat inositol phosphates and phosphoric acid. LITERATURE REVIEW Phytase enzyme Phytase (myo-inositol hexaphosphate phosphohydrolase, E.C.3.1.3.8) is the enzyme capable of hydrolyzing myo- inositol hexakis-dihydrogen-phosphate to yield inositol and free orthophosphate via inositol penta- to monophosphates as intermediary products. Phytase was the first enzyme known to liberate inorganic phosphate from organic phosphorus compounds (Suzuki et al., 1907) and as such has widespread distribution in plant and animal tissues, in many species of fungi and in certain bacteria (Cosgrove, 1966). Though most of the dry seeds contain phytate, the presence of it is not necessarily associated with phytase activity. There has been reported no phytase activity in cats (McCance and Widdowson, 1944) and mung beans (Mandal and Biswas, 1970), moderate phytase activity in barley (Preece and Grav, 1962) and high activity in wheat (Peers, 1953). The distribution and quantity of phytase are not in proportion to phytic acid content in seeds and not correlated with glycerophosphate and pyrophosphate activities in plant tissues (Courtois and Perez, 1948 a; Saio, 1964). During sprouting, all seeds possess phytase activity which increases with the progress in germination and is accompanied by an increase in the inorganic phosphate and decrease in the 3 4 phytate content of seed (Courtois and Perez, 1948 b; Peers, 1953; Mayer, 1958; Mandal and Biswas, 1970). Phytase activity was shown to be present in germinated pulses but not in ungerminated pulses, while phosphatase activity was found present in both germinated and ungermi- nated pulses. However, germinated pulses showed greater phosphatase activity (Belavady and Banerjee, 1953). Courtois and his collaborators have carried out extensive work on the occurrence, specificity and mechanism of phytase (Fleury and Courtois, 1945; Fleury and Courtois, 1947; courtois, 1947a; courtois, 1947b; courtois and Joseph. 1947; Courtois, 1948; Courtois and Perez, 1948a, 1948b; Courtois and Joseph, 1948; Courtois and Perez, 1949; Courtois, 1951; Barre et al., 1956). They observed that phytase behaves as a distinct enzyme different from the majority of other phosphomonoesterases. They noticed that phytase hydrolyzed inositol hexaphosphate whereas glycerophosphatase prepara- tions were inactive towards inositol hexaphosphate, but could hydrolyze lower phosphate esters of inositol (Courtois, 1945). Later they found that the phytase from wheat bran was active on both phytic acid and glycerophosphate whereas a common phosphatase associated with the phytase was inactive on phytic acid but active on glycerophosphate (Fleury and Courtois, 1947). They applied to the wheat bran the custom- ary techniques of separation and purification of enzymes but could not single out any evidence for the existence of a phytase that hydrolyzes specifically only the phytic acid (Courtois, 1947a). They concluded that wheat bran and mustard seed, two of the materials with which they worked most of the time, contain two distinct enzymes: a common phosphomonoesterase capable of hydrolyzingdkglycerophosphate but not phytic acid and a phytophosphatase (phytase) capable of hydrolyzing both substrates (Courtois, 1947b; Courtois and Joseph, 1947). Gibbins and Norris (1963) distinguished two enzymes in Dwarf french bean, the one being active towards phenyl phosphate but not towards phytate, and the other was active towards both phytate and phenyl phosphate. The first was an acid phosphatase and the second a phytase. Attempts to purify the phytase enzyme have been proved quite tedious. Nagai and Funahashi (1962) purified the wheat bran phytase more than 1500 times. The purified preparation was not a phytate specific phosphatase, but had all the characteristics of a nonspecific acid phospho- monoesterase with broad substrate specificity to various phosphomonoesters at pH 5.0. Their preparation had a potent pyrophosphate activity which is characteristic of plant nonspecific acid phosphomonoesterase. Peers (1953) found the phytase enzyme to be more dispersed throughout the wheat kernel than its substrate, phytate, yet the enzyme was found primarily in the aleurone (39.5%), endosperm (34.1%) and scutellum (15.3%). He also reported that enzyme activity was higher in hard wheats than soft but the variation in activity among the species was not large. Both enzyme and substrate found in the endosperm have been associated with protein bodies (Morton and Raison, 1963). The action of phytase on phytates is by steps as it is shown below (Sloane-Stanley, 1961): myoinositol hexaphosphate + H20-4»myoinositol pentaphosphate+ (1P6) (1P5) H3PO4 “’1’ followed by IP5 + H20«———>-IP4 + Pi’ etc. The stepwise reaction has been proved by Mihailovic and co-workers (1965) who found that wheat phytate was completely decomposed within seven days of germination. Using paper chromatography, they examined extracts of wheat made at various stages of germination where they observed the formation of intermediate penta-, tetra-, tri-, di-, and monophosphates of myoinositol. In ripe wheat grain before germination, only inositol hexaphosphate was present. The stepwise hydrolysis is in agreement with the results of in vitro studies of the action of phytase preparations on phytic acid. With the use of paper chromatography, Preece and co-workers (1960) found hexa-, tetra-, and tri- phosphates in barley. Malt contained all the above esters plus the diphosphate, the presence of which suggests that degradation occurs. The failure to detect di- and mono-esters in barley is attributed to limitations of the method, or, if they are present, their amounts must be very small. de Boland et al (1975) found only hexa- _ phosphate in the mature seeds of corn, wheat, rice, soy- beans and sesame. Glass and Geddes (1959) found an increased level of inorganic phosphorus along with lower phytate levels in wheat stored under elevated temperature and moisture conditions. Chemistry of phytic acid The presence of a Ca-Mg salt of an organic phosphate in the aleurone layer of wheat endosperm was first re- ported by Pfeffer (1872). Winterstein (1897) later showed that a similar substance phytic acid extracted from the seeds of Indian mustard (Sinapsis nigga) gave myo-inositol and orthophosphoric acid after hydrolysis with hydrochloric acid. Later Michel—Durand (1939) stated phytic acid to be as ubiquitous in the plant kingdom as starch. The chemical designation of phytic acid is myo-inositol 1, 2, 3, 4, 5, 6-hexakis (dihydrogen phosphate). The name "phytic acid" has been used interchangeably in the lit- erature with the term "phytin" which more correctly refers to the mixed Ca and Mg salt of the acid. Phytic acid and its isomers are unique in nature for being the only biologically produced molecules containing six phosphate groups on adjacent carbon atoms. The structure of phytic acid has been controversial for some time. The more recent work of Johnson and Tate (1969) indicates that cereal grain phytic acid has the myo-inositol hexaorthophosphate structure suggested by Anderson(1914). Figure 1.--Anderson's structure Biochemistry of phytic acid Phytates are found in a wide variety of foods as was demonstrated in an early study of Averill and King (1926), who reported a wide range of phytate levels as influenced by variety and byproduct of numerous cereals and nuts. According to Earle and Milner (1938),phosphorus compounds found in seeds may be classified into four groups: phytates, phosphotides, nucleic compounds, and inorganic phosphorus compounds. Phytic acid is the principal form of phosphorus in many seeds; 60-90% of all the phosphorus in some seeds is present as phytic acid (Barre, 1956). Occurence Utilizing scanning electron microscopy, Pomeranz (1973) observed that in the case of barley, phytate is in the form of potassium and magnesium salts instead of the calcium-magnesium complex normally thought to be present in most other cereals. Phytic acid levels in 18 varieties of barley were found to range from 0.97 to 1.08% dry weight (Lolas et al., 1976). Makower (1969) reported that mature dry pinto beans contained approximately 1% phytic acid whereas immature beans contained about 0.13%. In addition, low levels of phytic acid were found in the ' pod at all stages of maturity. Walker (1974) reported that embryo development to maturity in phaseolus vulgaris requires approximately 36 days. Approximately 90% of the phytic acid was found between days 24 and 30. During germination 90% of the phytic acid was lost by day 10. Lolas and Markakis (1975) measured the phytic acid content of 50 cultivated varieties of P.vulgaris grown over a 2- year period and found a range of 0.54-1.58% of dry basis. _.—_.....— 10 In addition, they also noted that 99% of the total phytic acid was in a water soluble form. Anderson (19140) was among the first to identify ,phytate in corn. Later, DeTurk et a1 (1933) followed phytate levels in corn from pollination to maturity and observed that phytate was not present in the leaves, stems, tassels, or cobs of the plant and that phytate began to increase in the kernels approximately 3 weeks after pol- lination and increased to maturity. O'Dell et al (1972b) demonstrated that in corn approximately 90% of the phytate is concentrated in the germ portion as.compared to the endosperm and hull portions. Engle and Guinn (1959), in working with germinating cottonseed, noted the dephospho- rylation of phytate resulted in the accumulation of in- organic phosphorus. Wozenski and Woodburn (1975) measured the phytic acid level in four food-grade cottonseed products and found significantly higher phytate levels in products of glandless seeds than in products of glanded ones. Ashton and Williams (1958) found that phytate phos- phorus is gradually broken down into inorganic phosphorus during germination of cats and that no phytate phosphorus was present after 2 weeks of germination. During panicle emergence and up to the milk ripe stage, they found no phytate in maturing oats; however, at maturity approximately 60% of the phosphorus was in the form of phytate. Asada. and Kasai (1962) reported that during the early stage of 11. rice ripening a major portion of the myo-inositol was in the free state but at the end of ripening period most of the myo-inositol was in the phosphate ester form which represented approximately 80% of the total phosphorus in the product. Free myo-inositol and myo-inositol phosphate was found in the grains, leaves and stems, and roots of rice, but the low levels in the latter two portions com- pared to the grain level indicated that biosynthesis occured in the grains themselves from sugars. Kennedy and Schelstraete (1975) reported that phytic acid was primarily found in the outer layers of rice grain. Spe- cifically, 2% of the outside kernel was found to contain 23 times more phytic acid than the intact kernel, and removal of the outer 13% of the kernel resulted in an endosperm that contained no detectable phytic acid. Phytates in soy appear to be unique in that although associated with protein bodies, they appear to have no specific site of localization (Tombs, 1967). Lolas et al., (1976) have reported that the phytic acid content of 15 soybean varieties ranged from 1.0 to 1.47% on dry weight, representing between 51.4 and 57.1% of the total phos- phorus. de Boland et al., (1975) reported on the phytic acid content of several commercially available soy products. Soy meal had a level of 1.42%, flakes, 1.52% and isolate, 1.52%. Jennings and Morton (1963b), reported that the 12 initiation of rapid phytic acid synthesis in wheat could be correlated to the time of restriction of supply of water to the endosperm during maturation. Williams (1970) also investigated the effect of water stress on phytic acid formation in maturing wheat and found similar results. O'Dell et al., (1972b) found a level of 0.32% phytate in the whole kernel of wheat with approximately 87% of it being associated with the aleurone layer, 13% in the germ, 2% in the endosperm, and none in the hull portion.h Morris and Ellis (1976) have reported that most of the phytate associated with wheat bran is in the form of mono-ferric phytate, which in turn is probably bound to cationic sites of proteins or other cellular components. In evaluating 38 wheat varieties, Lolas et al., (1976) found a phytic acid range of 0.62-1.35% (dry weight) in whole kernels, whereas the bran portion had phytic acid levels ranging from 4.59 to 5.52%, demonstrating that foods containing added wheat bran could have very high phytate levels. Figure (2) shows the longitudinal section of wheat kernel. Physiological Roles. Several physiological roles have been suggested for phytic acid in plants. Phytic acid has been generally regarded as the chief storage form of both phosphorus and inositol in almost all seeds. Hall and Hodges (1966) at- tempted to obtain an overall description of phosphorus 13 ”on C." cm“ In“ Ore-can “one Que-In ‘NDOS'IRM .o hon-o low-- Cello-en I.“ .0 Com AIn-ou €ch ‘ Lon: fluellnr 'uuo he. Cu. Inn-I > IRAN C'Iu Com - 'I.' Corn -2:' . [5.15 sum-.- R Duo-no II..." loll I... “In. logo to. ooooooooooooooooooooooooooo Figure 2.--Longitudinal section of wheat kernel (Baking science and technology) 14 metabolism associated with the germination of cats. Their results confirmed that phytic acid represents the primary storage form of phosphorus, about 53% of the total phos— phorus. ,Biswas and Biswas (1965) proposed that phytic acid represents an energy store. Sobolev and Rodionova (1966) reported that phytic acid was synthesised by a mixture of aleurone grains and mitochondria isolated from ripening sunflower seeds, when myo-inositol and succinate were present. They considered the process of phytin formation by the aleurone grains as an important link in the general chain of reactions leading to quenching of the physiological activity of the seeds during ripening. They further proposed that the biosynthesis of phytates is not associated with the utilization of hexose phosphate or glycolysis products but is due to the stepwise phos- phorylation of inositol. Williams (1970) presented evidence that phytic acid serves only as a source of phosphorus and cations for the germinating seed. He suggests that the synthesis of strongly chelating phytic acid exerts an effect on the cellular metabolism by com- bining with multivalent cations. He further states that these multivalent cations play a significant role in the control of many cellular processes, particularly those involving phosphotransferases on which energy metabolism depends. Asada and co-workers (1968) found that phytate 15 contains over 80% of the total phosphorus of mature rice grain and the turnover of phytate phosphorus is practically nil in the resting grain. From that they concluded that phytate is the final product of phosphorus metabolism in the ripening process. Samotus (1965) suggested that for- mation of phytic acid in seeds and tubers is a means of preventing the accumulation of excessively high levels of inorganic phosphate. He proposed the following mechanism of phosphorus distribution in potato tuber. Inorganic phosphorus penetrates into the tuber during plant growth; a part of this phosphorus is engaged in metabolic trans- formations and the remainder is bound in the form of phytin and phosphostarch. Support of the above claim is provided by the observations of Asada and Kasai (1959) who found an enhanced accumulation of phytic acid, relative to other phosphorus compounds in rice grains, upon increased applications of phosphorus fertilizer to the rice plant. Proteinephytate complexes Insoluble complexes are formed between protein and polyphosphates. When polyphosphates, such as phytic acid are added to protein solution below the pH of their isoelectric point, precipitation occurs, and the extent of the reaction is controlled by the pH of the system. Phytic acid forms salt-like linkages with basic groups on the protein molecule such as those of arginine, lysine 16 and histadine units (Cosgrove, 1966). Myers and Iacobucci (1974) suggest that charged carboxyl groups are a major factor in explaining the binding behavior of phytate to glycinin between pH 3.0 and 4.0. Calcium ion has been shown to have an effect on phytate binding to glycinin. Okubo and co-workers (1974) assume that calcium ion medi- ates phytate binding to glycinin above the isoelectric point, and both soluble and insoluble complexes can be formed. They give as a possible explanation of the binding that calcium ion acts as a bridge between the carboxyl groups of the protein and the phosphate groups of phytate. Okubo and co-workers (1976), working with glycinin, a major globulin of the soybean, showed no binding above the isoelectric point of pH 4.9. The extent of binding was found to increase with decreasing pH, from a value of zero at the isoelectric point to a maximum value of 424 equivalents of phytate per mole glycinin dimer (360,000 daltons). They also showed that calcium ions promoted dissociation of phytate—glycinin complexes at pH 3 and they contributed this to the competition between Ca2+ and the cationic sites of the protein for the phosphate groups of phytate. At pH 3.0, a 105 fold equivalent excess of calcium with respect to the protein cationic groups was necessary to completely dissociate the complex. Saio and co-workers (1967) studied the effects of protein-calcium- phytic acid relationships on the solubility characteristics 17 of soybean meal protein and found that the combinations among proteins, calcium and phytic acid are very labile in the alkaline range above pH 8.0, especially by heating. The same workers (1968) also found that phytic acid af- fects the binding of calcium by a cold insoluble protein fraction of soybean meal. Elimination of phytic acid from soybean meal extracts is considered an essential prelimi- nary step to the study of the individual soybean protein (Smith and Rackis, 1957). Wang (1971) described changes in the isoelectric focusing behavior of soybean whey protein caused by the addition or elimination of phytate which influences the net charges of proteins. It has been shown that phytic acid exhibits an inhibitory effect on the peptic digestion of ovalbumin and elastin (Barre, 1956). This effect is related to its property to form insoluble combinations with proteins, below their isoelectric point, in an acid medium, and in a range of pH which corresponds precisely to the optimum for the action of pepsin. Destruction of phytic acid during bread making Phytic acid is hydrolyzed during the breadmaking process. Consumption of whole wheat breads that have been made with little or no fermentation has caused consider- able concern because of their high phytate levels (Reinhold 1971, 1972). Reinhold (1971) suggested that 18 yeast might contribute in decreasing the phytic acid content of leavened bread. Yeast or sourdough fermenta- tion of dough has been shown to lower phytate levels by one-third to half (Reinhold, 1972). Earlier, Mellanby (1944) had demonstrated that fermentation time, temperature, pH, and humidity could all significantly influence phytase activity. Reinhold (1975) reported that phytate loss due to the action of phytase was rapid in fermented bread made from 75-90% extraction wheat but was slow for 95-100% extraction products. de Lang et a1 (1961) reported that phytate is completely hydrolyzed in low extraction flours. Harland and Harland (1980) showed a significant reduction in phytate contents by increasing the amount of yeast and time of fermentation in rye, white and whole wheat breads. In contrast, Tangkongchitr et al (1981b) did not find a significant loss of phytate with higher yeast levels. Ranhotra (1972) reported 20 times more phytic acid in wheat protein concentrate than wheat flour, and thus the inclusion of wheat protein concentrate into bakery items can present phytate-related problems inspite of the fact that a high level of phytase activity is associated with it. Addition of calcium has shown to inhibit phytate hydrolysis in bread making (Ranhotra, 1972), and thus additives high in calcium content, such as certain whey products, can diminish phytase activity (Ranhotra, 1973). Ranhotra et al (1974) found little 19 phytase activity in a number of commercially available soy products. They also showed that the addition of 10% soy protein concentrate to the bread formulation resulted in phytate hydrolysis in excess of 80%, in contrast to 22% observed in a whey-soy blend product, probably due to high residual level of calcium in the latter blend. Knorr et al., (1981) found a reduction of upto 1/8 and 1/2 of the initial phytate concentration, upon addition of com- mercial phytase and phosphatase respectively. They also reported that storage of whole wheat bread for up to 96 hrs at room temperature further reduced phytate phosphorus. Nutritional aspects of inositol phosphates Ruminants are able to utilize phytates. Ellis and Tillman (1961) investigated the availability of phosphorus in wheat bran fed to sheep. They found appreciable amounts of phytin to have been digested. Rumen micro organisms show high phytase activity and evidence exists that the hydrolysis of phytates is due to the phytase of these organisms and is not dependent on phytases present in the feed (Raun et al., 1956). Non-ruminants do not/seem to be able to utilize phytates although phytase has been shown to be present in the intestinal mucosa of rats (Pileggi, 1959) and in human faeces, the latter being possibly bacterial in orgin (Courtois and Perez, 1949). The ability of man to hydrolyse 2O phytates remains a controversial subject, though some hydrolysis in the digestive tract occurs probably due to microbial phytases or non-enzymatic cleavage. Evidence has been presented recently that man probably possesses phytase (Bitar and Reinhold, 1972), but the lack of phytate cleavage may be caused by inhibitors of phytate hydrolysis present in foods such as bread (Reinhold et al., 1973). Phytases can only act on phytates in solution and the extent to which phytates are hydrolyzed depends largely on their solubility. This in turn depends on the ions with which they are associated and on the level of calcium in the diet. Zinc deficiency was first recognized by Prasad et al., (1963) in Egyptian boys whose diets consisted mainly of bread and beans. These patients, who were characterized by dwarfism and hypogonadism, showed a response to zinc supplementation. Erdman (1979) reported that the greatest impact of phytic acid relative to human nutrition is its reduction of zinc bioavailability. Likuski and Forbes, (1964, 1965) showed an inverse relationship between the level of phytic acid in the diet and zinc bioavailability. Forbes and Parker (1977) demonstrated that zinc added to rat diets in the form of whole fat soy flour was signifi— cantly less utilized than zinc added as zinc carbonate to an egg white diet. In the case of soy-fortified wheat bread, Ranhotra et a1 (1978) found that the bioavailability 21 of zinc was not affected because most of the bound zinc was liberated due to the action of phytase on phytate during fermentation. Momcilovi and Shah (1976b) studying the bioavailae; bility of.zinc to rats from several infant formulas and breakfast cereals concluded that the infant cereal was the poorest source of zinc. Reinhold et al., (1973a,b), utilizing human subjects found a positive correlation between the phytate levels and unavailability of zinc. The interaction of zinc, calcium and phytic acid has also been investigated by using the pig (Oberleas et al., 1962) and rat (Oberleas et al., 1966) as models and found that high levels of calcium in conjunction with phytate decreased zinc bioavailability. Iron represents the other nutritionally significant mineral that has been associated with phytate binding. Although there is little doubt that the consumption of) a diet containing added phytate (Na) lowers the iron balances in human subjects (Turnbull et al., 1962), the effect of phytate naturally present in foods has been reported to have no effect or only slightly depressing effect on the utilization of iron by rats (Ranhotra et al., 1974). This has been questioned by Davis and Nightingale (1975) who reported a significant effect. Morris and Ellis (1976) isolated monoferric phytate from wheat bran which they found to be water sobuble and of 22 high biological value to the rat. They also postulated that the monoferric phytate in bran was bound to calcium sites of proteins with utilization being through solu- bilization by an ion-exchange type mechanism instead of through phytate hydrolysis. Definite evidence that the presence of phytates in diets cause a reduction in copper availability has been obtained by Davis and Nightingale (1975) in studies with rats. Davis et al., (1962) had earlier reported that diets containing an isolated soy bean protein reduced the availability of copper in chickens. However, and in view of the high phytate content of soybean meal (Common, 1940) and the ability of soybean protein to complex with phytates, it would seem likely that phytates are involved in copper unavailability. Anticalcifying and rachitogenic properties were attributed to consumption of cereals by Bruce and Callow, 1934; Harris and Bunker, 1935; Cruickshank et al., 1945; Walker, 1951. In the case of dogs Mellanby (1949) demonstrated that phytate addition to their diets reduced calcium absorption and subsequently induced rickets. In contrast, Walker et al., (1948) reported that human diets high in phytates improved the retention of dietary calcium and magnesium. However, in a later study, Reinhold et al (1973b) could not confirm human adaptation to high- phytate diets. 23 Forbes (1964) reported that in rats, dietary calcium depressed weight gain, feed intake and femur zinc concen- tration, especially in the presence of soy protein. Likuski and Forbes (1965) showed that dietary calcium and phytic acid also decreased magnesium absorption. Van Den Berg et al., (1972) studying the influence of phytic acid and its derivatives on inhibiting calcification in the rat found that although phytate itself was inert, phytic acid and its hydrolysates were potent inhibitors of calcification. In the case of wheat breads, Reinhold et a1 (1975) contended that fiber and not phytate is primarily respon- sible for poor calcium absorption. They reported that the ability to bind calcium is a function of fiber con- centration. Evans and Pierce (1981) isolated a calcium-phytate complex, the chemical composition of which indicates penta substituted calcium phytate, despite rather widely varying P/Ca ratios in the reaction mixture, Earlier, Hoff-Jorgensen had previously (1944) reported penta- calcium phytate. Evans and Pierce (1982) studying the simultaneous interaction of several metal ions with phytic acid could isolate only amorphous powders with non- stiochiometric atomic ratios, however, Hay (1942) reported a hexa-calcium phytate salt in the corn. MATERIALS AND METHODS Enzyme extraction The crude enzyme solution was prepared according to (Lolas and Markakis, 1977) with some modifications’from baker's yeast cake. The yeast cells were hydrated with distilled water for 30 min, broken by Polytron (Brinkmann Instruments, Inc., Westbury, New York) and extracted with a 10:1 ratio of 2% CaCl to yeast. The enzyme 2 solution was centrifuged at (2000 g) for 30 min at 20 C. The clear supernatant solution had a pH of 5.3. Ammonium sulfate fractionation Sufficient solid ammonium sulfate was added to the crude enzyme solution (Dixon, 1953) with continuous mechanical stirring to make it 30% saturated. The enzyme solution was kept for 30 min at 2°C and centrifuged (2000 g) for 20 to 30 min at 2°C. The residue was dis- carded and the supernatant solution was made 80% (NH4)2S04 saturated followed by the same treatment as above. The fraction precipitating between 30% and 80% saturation contained all the phytase activity. This was dissolved in a small volume of 0.01 M tris-maleate buffer pH 6.5 and dialysed for about 48 hours in the same buffer in a 24 25 cold room (2°C). Assay procedure Phytase activity was assayed by measuring the rate of increase in inorganic phosphorus, liberated by the action of phytase, using the ascorbic acid method (Watanabe and Olsen, 1965). The reactions were carried out in small glass-stoppered test tubes in a 45 : 1°C water bath. The typical reaction mixture had a total volume of 1.2 ml and contained 0.2 ml of 0.6 M acetate buffer, pH 4.6; 0.15 ml of 8 mM sodium phytate (SIGMA Chemical Co; St. Louis, MO) previously adjusted to pH 4.6 (with 1 N HCl), 0.2 ml enzyme solution and water to 1.2 ml. Final concentration of buffer and phytate were 0.1 M and 1 mM, respectively; and incubation time usually 30 mins. After incubation, samples were withdrawn from the digest, deproteinized by adding 0.8 ml of 10% TCA, cen- trifuged in small 2 ml conical centrifuge tubes and orthophosphate determination was carried out on the supernatant according to the method described below under the title "Determination of inorganic phosphorus by the ascorbic acid method". The activity values were corrected from a control which contained boiled enzyme. Enzyme activity was expressed in international units, one unit being the activity which results in the libera- tion of 1/umole of inorganic phosphorus per minute --- 26 (recommended by the Commission on Enzymescufthe Inter- national Union of Biochemistry) (Whitaker, 1972). Determination of inorganic phosphorus by the ascorbic acid method The steps of the method are as follows: 1. Prepare reagent A. Dissolve 12 g of ammonium molybdate in 250 ml de- ionized H20. In 100 ml of deionized H 0 dissolve 0.2908 g 2 of antimony potassium tartrate. Add both of the dissolved reagents to 1 liter of 5 N H2804, mix throughly, make to 2 liters and store in a brown glass bottle in the refrigerator. 2. Prepare reagent B. Dissolve 0.264 g ascorbic acid in 50 ml of reagent A and mix. This reagent does not keep more than 12 hours and must be prepared before analysis. 3. Pipette aliquots containing 0.01 to 0.015lumole of orthophosphate into 5 ml volumetric flasks. 4. Add deionized H 0 to make the volume to 4 ml, 2 and then add 0.8 ml reagent B. 5. Make to volume with deionized H20 and mix. The color is stable in 10 min and is measured at 700 nm. 6. A standard curve was prepared using a standard phosphorus (predried KH P04) solution in the same manner 2 as above against a blank containing 4.2 ml H20 and 0.8 ml 27 of reagent B. The following linear regression equation was used for estimating the Pi content A = 0.003 + 700 3.204 x C. The correlation coefficient corresponding to this equation was (r = 0.989). Effect of pH on enzyme activity Standard assay procedures were used to determine reaction rates over the pH range 3.6 to 6.0. Acetate buffers were used except for pH 6.0 where a tris-maleate buffer was used. The buffers had a final concentration of 0.1 M in the assay mixture. The results were expressed as percent of activity at pH 4.6 and plotted against pH. Effect of incubation temperature on reaction rate The reaction rates were determined at temperatures from 40°C to 60°C at 5 degree intervals using standard assay procedures. The inorganic phosphorus liberated was measured after 30 min of incubation. Effect of substrate concentration on reaction rate The effect of phytic acid concentration (final concentrations up to 10 mM) on activity was tested by measuring initial reaction velocities. The Michaelis constant and maximum velocity values were calculated by plotting 1/(initial velocity) against 1/(substrate 28 concentration) (Lineweaver and Burk, 1934). For the determination of Km, the initial velocities of reaction were measured over the range 0.05 to 8.0 mM phytate. Effect of various metal ions on phytase activity Ca2+ (CaS04.2H20); Cd2+ (CdCl .2%H20); €02+ (CoCl 2 6H20); Cu2+ (CuSO4.5H20); Fe2+ (FeSO 2. 2+ 4.7H20); Hg (HgClz); 2+ 2+ 2+ Mg (Mg (N03)2.6H20); Mn (MnSO4.H20); Zn (ZnSO4. 5M, 10‘4M and 7H20) ions at final concentrations of 10- 10-3M were investigated to determine their effect on enzymatic activity. These ions were incorporated into the assay by the addition of each separately and the results were checked against those of controls containing no metal ion. Effect of chelating agents on phytase activity Standard assays were performed which contained ethylenediamine tetra-acetic acid (EDTA), sodium oxalate, sodium potassium tartrate and sodium citrate at final concentrations of 10-6M, 10_4M, and 10-2M. The results were compared with those of controls containing no chelating agent. 29 Separation of phytase from phosphatase Diethlaminoethyl (DEAE) cellulose (20g) was treated according to the procedure described by Whitaker (1972). It was packed in a glass column (3.0 cm x 50 cm) to a height of about 27 cm. The column was equilibrated with 0.01 M tris-HCl pH 7.4 buffer, until the pH of the efflu- ent was identical with that of the applied buffer. The enzyme solution was dialysed against the same buffer for 24 hours and about 30 ml of enzyme solution, having a protein content of 3.3 mg/ml, was charged gently at the top of the column. The concentrations of NaCl solution in 0.01 M tris-HCl buffer, used for elution of the protein from the column, were 0.15 and 0.40 M (stepwise method). The 0.4 M NaCl concentration was applied after all the phosphatase enzyme had been eluted. The chromatographic procedure was carried out at room temperature. The collected fractions (10 ml) each were assayed for protein content, phosphatase and phytase activity. The assay for the phosphatase activity was carried out under the con- ditions as that of the phytase enzyme. For the determination of protein the spectrophoto- metric method of Warburg and Christian (1942) was used and the protein content calculated by the Kalckar (1947) formula: 1.45 x (absorbance at 280 nm) - 0.74 x (absorbance at 260 nm) = mg protein/ml 30 Substrate specificipy The DEAE-cellulose separated phytase, after dialysis, was used to determine its activity against 5'- adenylic acid (AMP), d and P- glycerophosphate, sodium pyro- phosphate, phenyl phosphate, inositol pentaphosphate, inositol tetraphosphate, inositol triphosphate, inositol diphosphate, inositol monophosphate and phytic acid.. The intermediate inositol phosphates were obtained from the wheat extracts. The activities of the above substrates were compared with that of phytic acid. Standard assay procedures were used with the only difference that all the substrates, except phytic acid and the intermediate inositol phosphates, were added in the assay mixture at a final concentration of 10 mM. Preparation of bread Two experiments involving bread were conducted. Variable milling extraction rate was the factor studied in the first one, and variable fermentation time was studied in the second experiment. A. Soft Red Wheat (SRW) flours of 70%, 80%, 90% and 100% extractions were obtained from Soft Wheat Quality Laboratory, Wooster, Ohio. The breads were made according to AACC-10-10 procedure (1962). The formulation of test pan bread were: flour: 100 g ; yeast: 3.0 g; salt: 1.5 g; sugar: 5.0 g; and water: 70 ml. The yeast was first 31 hydrated in a mixing bowl for 5 min and then sugar, salt and flour were added to it. The ingredients were first blended at low speed for 1 min and then the dough was mixed at high speed for 7 min in a Hobart mixer. The doughs were fermented for 120 min at 30°C and 85% R.H. The dough were given a 10 min bench rest after fermenta- tion at room temperature. The loaves were then molded, panned and later proofed for 40 min at 30°C and 85% R.H. The proofed doughs were baked at 218°C for 20 mins. The breads were air dried, ground and stored in a dessicator for later use. Inositol phosphate analysis was conducted on the flours as well as on the breads made from them. B. The wheat flours (WF) and whole wheat flours (WWF) that were used in the fermentation experiment were obtained from a local market. All the breads were made according to AACC-10-10 procedure and with same formulation of test pan bread as used in extraction experiment, except the doughs were fermented for 30, 60, 90 and 120 min at 30°C and 85% R.H. The 0 min fermentation dough was immediately used for separation of inositol phosphates. The breads were later air dried and stored in a dessicator for later use. Commercial whole wheat and white breads obtained from local market were also analysed for the entire spectrum of phosphates. 32 Separation of inositol phosphates Separation of inositol phosphates was done according to the method of Saio (1964). The inositol phosphates were extracted from 1 g of wheat flour or bread sample with 10 ml of 3% TCA. After centrifugation at 12,000 x G, 1 ml of the extract was chromatographed on a Dowex 1 x 8 (200-400 mesh, Cl- form, 1.1 x 11 cm) column. The extract was eluted with 600 ml 0 - 1.0 N HCl linear gradient at a flow rate of 2 ml per minute. Eluant was collected in 120 tubes (5 ml per tube) using a fraction collector (Rinco Instruments Co., Greenville, ILL). Phosphorus determination of chromatographic fractions The solution in each fraction was evaporated to dryness at 40°C by blowing air onto the surface of the solution through a manifold (Figure 3). 1 ml of 70% perchloric acid was added to each fraction containing dry residue. The tubes were heated for 45 min to release the phosphorus from the inositol phosphates. The phosphorus content of each fraction was then deter- mined colorimetrically according to Allen's method (1940) with slight modifications. The method was based on the formation of phosphomolybdic acid which was reduced to an intense blue complex. Reagents used in phosphorus determination were as follows: Perchloric acid: a 70% solution 33 m3 .moQSu cw casuda mcwaononm>o new mom: maumsmqaHH>«uon encased «new» on» so codumuunoosoo dungeonsm no poemHMIi.o unawam SE .2028 mimpmgm o. o w a o [I 4” 1 q . no. 6 2N3 1W/NIWI!d w" '31sz Nouovaa 47 enzyme units/ml enzyme, 5 = substrate concentration as molarity) the Michaelis constant Km was found to be 0.21 mM and the Vmax 0.005 umoles/min per ml enzyme (Figure 7). Other Michaelis constants for phytase reported in liter- ature are 0.091 mM for corn phytase (Chang, 1967); 0.33 mM for wheat phytase (Peers, 1953); 0.57 mM for wheat bran phytase (Nagai and Funahashi, 1962); 0.15 mM for the Dwarf bean phytase (Gibbins and Norris, 1963); 0.65 mM for the mung bean phytase (Mandal et al., 1972); and 0.018 mM for the Navy bean phytase (Lolas and Markakis, 1977). Effect of metal ions on phytase activity As shown in Table 1, none of the metallic ions tested including magnesium and calcium had any effect upon phytase activity with the exception of cadmium, mangnese, mercuric ions that have inhibitory effects at final concentration of 10‘3M and higher. This inhibitory effect of metallic ions may be due to strong affinity to phytic acid itself and resulting in some competition between the metallic ions and enzyme for the substrate. Obviously, heavy metallic ions can have an adverse effect on the enzyme protein. Peers (1953) reported activation of wheat phytase by magnesium and calcium ions. Gibbins and Norris (1963) 48 P (I .Aoa>us0 Ha son :Ha\woaoa=\sH commohaxo muH00H0>v .mumnumnsm mm ounpzna sawoom and? outpusa ammo» mo “GHQ xssmuso>noaocaqiizh ossmfim 2E.:0Hussu:oosoo m\p Mm WIN A ..e . s m 3. Mb 0'" AltooIaA/L co -I\ 49 Table 1.--Effect of metal ions on the yeast phytase acitvity. ‘_—"""""""""—'—"'—'_"""——‘""T““‘If""""""‘ Relative Activity Metal ions 10'5 ‘4 M ‘3 M Caso4 99% 100% 106% CdC12 102 79 72 00012 101 101 94 Cuso4 95 116 115++ Feso4 105 125 130++ HgCl2 100 80++ 66++ Mg(N03)2 94 91 97 Mnso4 109 90 87++ Znso4 103 103 97++ @The activity without added metal ions was taken as 100%. ++Precipitation was observed. also observed a small increase in the activity of the Dwarf bean phytase by the same metals whereas no activa- tion was observed in the bran wheat phytase (Nagai and Funahashi, 1962). Lolas and Markakis (1977) showed an increase of 35% in activity of Navy bean phytase by cobalt in the enzyme mixture. The yeast phytase, in the present study, showed an increase in activity by about 30% as a result of the addition of 10-3M Fe2+ in the enzyme mixture. There is no data in literature on the 50 effect of Fe2+ on phytase activity. However, a sample of wheat phytase (SIGMA Chemical Co., St. Louis, Missouri) when assayed did not show any effect in the presence of 10‘3M Fe2+. Effect of chelating_ggents on yeast phytase activity As shown in Table 2, all the chelating agents tested showed a decrease in the phytase activity. In the presence of EDTA, even at the low concentrations of 10-6M, the activity of yeast phytase decreased by 72%. This behav- iour of the yeast phytase may be due to the fact yeast phytase is activated by Fe2+, and the presence of chelating agents in the reaction mixture may result in non- availability of Fe2+ for the activation of the enzyme. Table 2.—-Effect of chelating agents on yeast phytase activity. Relative Activity@ Chelating Agent 10’6 M 10‘4 M 10'2 M Citrate 100% 68% 50% Oxalate 80 64 47 Tartrate 93 90 39 EDTA 72 66 40 @The activity without added chelating agent was taken as 100%. 51 Separation of phytase from phosphatase Figure 8 illustrates the fractionation of a crude enzyme extract from yeast phytase on a DEAE column. The first enzyme emerging from the column is a phosphatase that hydrolyzes a<-glycerophosphate 0r phenyl phosphate but not phytic acid. The second enzyme is the phytase which utilizes all the above substrates. As a further proof of the different nature of enzymes is the fact that the phytase fraction,but not the phosphatase fraction showed an increase in the activity on phytate by about 30% in the presence of 10-3M Fe2+. Thus it is evident that a clear distinction can be drawn between the two enzymes. Substrate specificity of yeast phytase The yeast phytase purified by DEAE-cellulose chroma- tography, had a broad sutstrate specificity, catalyzing the hydrolysis of all the phosphomonoesters tested Table 3. Yeast phytase hydrolyzes all the substrates tested and is characterized by a potent pyrophosphate activity. Among the substrates used, pyrophosphate was the most preferred substrate while inositol pentaphosphate was the least preferred substrate. The enzyme was most active on inositol monophosphate, followed by inositol triphosphate, inositol diphosphate, inositol hexaphosphate, inositol 52 .mpa>fiuos compass new games one use ommumasmoad non ha sopsmo 0:» ca mason puma one .>9H>Huom onsuhnm . .>HH>Huos ommunsamonm . "as cam on oocmnaomns . .v.> =9 .sommzn Homnmume a Po.o as sodasaom Homz suds essaoo song cognac was cannons mmmzsz zo~so u m: mucosa pawns odes; n mg; n .056amoh HomeocH mom mopmnmmozm m 09 F was a Issuance mopmnmmosn Heuumocw u AH 0» AH "monogamosm cacomaosa u A © rpm mm For ov mop we on opp m3 0mm mm Pup map NPF our NPP hop as: our own no opp mm Pb no on be m: vmm mmp mmp om omp NNP as war m3: om Ppm For mm Pm mm Pb mm vs m: Now new mop NPF oop vow mm opp m3: om vpm mp? mm an no mop mm on m3 mmm wow nor am hNP mpp mm mm m3: om mpm Nap no mm vm mm mm ov m: how mam Far mm mm so so me m3: 0 seduces“ o no monogamonn acaqmoca cu musopcoo snows snoHH am0:311.® canoe .Amqmmn zpo .Unosn no w cop \mc0auomhn one we soon ca “commas n no msv mafia codenHGQEAom no 61 .0mm h AsoHHsm one comes so w as an sacs caused no codesasuozdmonnoo Ho owonwmuu.op magmas ma .. so 3 3 K 3 3 3:32. 1 3.04 0.5... K 4' 3 k .3 3 3 .3 3 as no as 3 .. g A. g o 3 1.3 h 1.3 , 3 3 3 h 3 3 1 to 62 the figures pertaining to phytate destruction in bread- making vary greatly: from 100% for white bread (Pringle and Moran, 1942), to 40 - 50% for whole wheat bread (deLange et al., 1961), to 13% for village flat breads made in Iran (Reinhold, 1972). As the phytate content decreased, the inoraganic phosphate content increased (Figures 11 h 12). The largest increase in P1 in WWB occurred during the last 30 minutes of fermentation. This may be due to the activity of wheat phytase in the earlier stages of fermentation, as is present in large amounts in whole wheat flour and yeast phytase activity in the later stages of fermentation. But not even after 120 minutes of fermentation can the loss of IP6 be accounted for by the Pi rise. Only when the P content of the intermediate inositol phosphates are taken into consideration can a acceptable P balance be achieved. Tongkongchitr et al., (1981) could account for almost all of the phytate P loss by the increase in inorganic P, but this occurred after 8 hours of fermentation by which time perhaps almost all of the intermediate inosi- tol phosphates were dephosphorylated. Such prolonged fermentation time would lead to lowering of loaf quality and a decrease of the nutrient content of bread (Reinhold, 1975). The intermediate inositol phosphates content of breads fluctuated with fermentation time, which is probably due to several dephosphorylation reactions / 100 g bread .1 mg P. 63 130 ~ *5 500 160 - if / f / I40 1‘ ,1 \ / I - 350 \ l’ 120 - \ / \ / x 100- \ I \ P/ K 80 ~ .I I" \ \ 4 200 / ‘ x I ‘n\ \ \ \ 60 r I \ ‘ ‘R I \\.\ t ‘ \ - PHYrI-c 4° ACID 1. ‘50 I Al 4 n "o 0o 30 60 90 120 FERMENTATION TIME, MIN Figure 11.-—Effect of fermentation time on the phytic acid and Pi content of whole wheat bread. mg phytic acid / 100 g bread / 100 g bread .1. mg P. 64 110' pp. ~200 I \ I I 100- \ / \ / I \ / - \ -150 1 \ 80 r \ \a. \ A q I \ \R / . I . \ 2' 100 / / ,c/ \ , ’ \ 60 _ ’ z I \U\ Pr I \ / \ I \n ' I ’ PHYTIC ‘50 , ACID / 4 / I ' f o 30 60 90 120 FERMENTATION TIME, MIN Figure 12.--Effect of fermentation time on the phytic acid and Pi content of wheat bread. mg phytic acid / 100 g bread 65 occurring simultaneously. Inositol phosphates in commercial breads The inositol phosphate contents of commercial breads are given in Table 7. The white bread shows more P IP 1’ 6 and total phosphorus than expected, assuming a 70 - 75% extraction. The excessive amounts of Pi and total phos- phorus must be due to the added mono-calcium and di-calcium phosphates listed on the label of the white bread. The rather large quantity of phytic acid may be attributed to the inhibition of the yeast phytase by the added calcium in the form of calcium propionate, mono-calcium phosphate, calcium sulfate and di-calcium phosphate; such inhibition was previously observed by Ranhotra (1972). The whole wheat breads showed only 17.4% of phosphorus in the phytate form, and other inositol phosphates did not show any pattern due to continuous phosphorylation and dephos- phorylation reactions. The whole wheat breads did not contain any added calcium or phosphates according to the label. INOSITOL PHOSPHATE-METAL BINDING Inositol monophosphate-metal complexes: IP1-meta1 complexes were formed at all the pH's studied except at pH 4 with zinc (Table 8). IP1-Ca precipitate showed phosphorus to 66 .asmocw> .pmoow .aHmm .wsfisouaozm cannuomo> .Q=s>m :uoo .:H>namon«m .maospacnocoa :Henqze .conm .swomaz .moanmn ocean: .Anson asossv sneam convened oosomoanca .umoss ossosw "mucoaooswca venom poms; macs; .:H>mHHonHm one .moasoHnoouohn casndne .mumcoH sawmmsuom .ounsonsso as 0N< .oHMHasm moossom .cdomaz .ounsoun ssflmmmaom .moshnco damask .mamwasm anacoas< .uHms hoasmm .oumnnmonq asaoamoHn .mamwasm ssfioamo .mumnmmong ESHOHcoocoz .ooasoomamosoz .0ud:0aaosg ssdoamo .ssoHH :aoo .xaqs ammlcoz .oamnnopm azaoom .pHmm .Hfio smonhom .pmwow .mon3 .Qshhm ssoo .nsoflm "mucofisoawsm cmosm been: .>H0>Hpooamos .osoamos Hepamocq sea masmsm ounmamozs a Cu P mcammommos « moacasmozd Heuamoca u QH ca mm.moumnmmonm cacomhosa u .m © sebum owe Pm so so no om om our onus: oHosz anqosossoo men we as F. on mp m cop snogm oases Hoaosoesoo a so no so me we Po so nosooge no fish .Amwmnn has .Umosn no m cop and seduces“ some ca Hammond A no mev mucosa anon; macs: one means @maouoesoo 02p :a moumnamonq casmmsosa use monogamonm Heuamosa no codamsunoo:0011.b canoe Table 8.--Percentagesof metal and phosphorus precipi- tated from Inositol monophosphate solution at pH's 4,5 and 6. P/metal in Metal % metal % phosphorus precipitate Ca (as CaC12) 30.01 23.25 1.0 31.28 28.52 1.17 33.68 31.07 1.00 Cu (as CuSO4) 9.62 14.08 2.99 62.04 30.27 1.00 95.61 8.40 @ Fe (as FeSO4) 50.16 13.92 0.500 77.10 14.26 0.333 93.22 6.55 @ Zn (as ZnSO4) — — — 35.50 8.41 0.500 38.58 4.40 @ @ These precipitates contain both metal hydroxide and inositol monophosphate-metal complex. No precipitate formed. 68 calcium atom ratio of 1 : 1 at pH's 4 and 6, while at pH 5 a ratio of 6 : 5 was observed. The IP1-Cu precipitate at pH 4 had a phosphorus to copper atom ratio of 3 : 1; at pH 5 the ratio was 1.: 1. The IP eFe precipitate 1 analysis showed a phosphorus to iron atom ratio of 1 : 2 at pH 4 and 1 : 3 at pH 5. The IP1-Zn precipitate displayed a phosphorus to zinc atom ratio of 1 : 2 at pH 5. As the pH increased there was an increase in the percent of metal precipitated. The influence of pH was more pronounced in the c0pper and iron precipitation than in the calcium precipitation. Increasing the pH from 4 to 5 resulted in a large change in the atom ratio of phos- phorus to COpper: from 3 : 1 to 1 : 1. For a similar pH increase the atom ratio of P : Fe changed from 1 : 2 to 1 : 3. Calcium did not show any change in the atom ratio of 1 : 1 with phosphorus as the pH increased. Inositol diphosphate-metal complexes: IP2—metal complexes were formed at all pH levels (Table 9). The IP2-Ca com- plex analysis showed a simple atom ratio of phosphorus to calcium of 1 : 1 at pH's 4, 5 and 6. The IP2-Cu precipitate showed a phosphorus to c0pper atom ratio of 2 : 1 at pH 4 and 1 : 1 at pH 5. The IP2-Fe precipitate, at pH 4 had a phosphorus to iron atom ratio of 1 : 1, at pH 5 the ratio was 1 : 2. The IP2-Zn complex displayed a phosphorus to zinc atom ratio of 1 : 2 at pH 4 and 1 : 1 at pH 5. 69 Table 9.--Percentages of metal and phosphorus precipi- tated from Inositol diphosphate solution at pH's 4,5 and 6. P/metal in Metal pH % metal % phosphorus precipitated Ca (as CaC12) 4 29.01 22.47 1.00 5 31.50 24.41 1.00 6 32.62 25.26 1.00 Cu (as CuSO4) 4 20.98 20.47 2.00 5 72.24 35.24 1.00 6 90.82 12.52 @ Fe (as FeSO4) 4 49.88 27.68 1.00 5 81.54 22.63 0.50 6 97.51 9.53 @ Zn (as ZnSO4) 4 17.23 4.08 0.50 5 34.72 16.45 1.00 6 49.54 4.52 @ @ These precipitates contain metal hydroxide and inositol diphosphate-metal complex. 70 All the metals showed an increase in the percent of metal precipitated with an increase in pH. The increase in IP2-metal complex precipitation was larger in copper, iron and zinc than in calcium. With an increase in pH from 4 to 5, the atom ratio of P : Cu decreased from 2 : 1 to 1 : 1. For a similar increase in pH, the P : Fe atom ratio changed from 1 : 1 to 1 : 2, while the zinc precipi- tation there was an increase in the P : Zn atom ratio from 1 : 2 to 1 : 1. There was no change in the atom ratios of IP2-Ca complexes with a change in pH. Inositol triphosphate-metal complexes: IP3-metal complexes were precipitated at all pH's (Table 10). IP3-Ca complex formed a simple phosphorus to calcium atom ratio of 1 : 1 at all the pH's studied. The IPS-Cu precipitate displayed a phosphorus to copper atom ratio of 2 : 1 at pH 4, and 1 : 1 at pH 5. The IP3-Fe precipitate analysis showed a phosphorus to iron atom ratio of 1 : 1 at pH 4 and 1 : 3 at pH 5. The IP3-Zn complex showed a phosphorus to zinc atom ratio of 1 : 2 at pH 4 and 1 : 1 at pH 5. The effect of pH on the IP3-metal complexes was significant in copper, iron and zinc, while calcium showed only a slight increase. All the metals showed an increase in percent metal precipitated with an increase in pH. Increasing the pH from 4 to 5 resulted in decrease in the atom ratio of phosphorus to copper from 2 : 1 to 1 : 1. Table 10.--Percentages of metal and phosphorus precipitated from Inositol triphosphate solution at pH's 4, 5 and 6. Metal pH % metal % phosphorus p/metal in precipitated Ca (as CaC12) 4 25.12 19.47 1.00 5 30.36 23.53 1.00 6 32.28 25.02 1.00 Cu (as CuSO4) 4 15.74 15.36 2.00 5 70.51 34.39 1.00 6 99.71 25.31 @ Fe (as FeSO4) 4 58.78 32.63 1.00 5 94.61 35.00 0.66 6 99.91 20.04 @ Zn (as ZnSO4) 4 9.46 2.24 0.500 5 42.91 20.34 1.00 6 50.20 21.06 @ @ These precipitates contain metal hydroxide and inositol triphosphate-metal complex. 72 For a similar pH increase the atom ratio of P : Fe in the IP3-Fe precipitate decreased from 1 : 1 t0 1 : 1.5, while the IP3-Zn precipitate showed an increase in the atom ratio phosphorus to zinc from 1 : 2 to 1 : 1. Calcium did not show any change in the atom ratio of 1 : 1 between phosphorus and calcium with an increase in pH. Inositol tetraphosphate-metal complexes: IP4-metal complexes were precipitated at all pH's studied (Table 11). The IP4- Ca complex showed simple atom ratio of phosphorus to calcium of 1 : 1 at the pH's 4, 5 and 6. The IP4-Cu precipitate analysis showed a phosphorus to copper atom ratio of 3 : 2 at pH 4, and 1 : 1 at pH 5. The IP4-Fe complex had a phosphorus to iron atom ratio of 1 : 1 at pH 4, and 1 : 3 at pH 5, while in the IP4-Zn complex the P : Zn ratio was 1 : 1 at pH 4 and 6 : 5 at pH 5. As the pH increased there was an increase in the percent of metal precipitated. The influence of pH was greater in the copper, iron and zinc precipitation than in the calcium precipitation. Calcium did not show any difference in the atom ratio of 1 : 1 with phosphorus precipitates as pH increased. The phosphorus to copper atom ratio decreased from 1 : 0.68 to 1 : 1 with an increase in pH from 4 to 5. For a similar pH increase the atom ratio of phosphorus to iron changed from 1 : 1 to 1 : 1.5, while P : Zn atom ratio changed from 1 : 1 to 73 Table 11.--Percentages of metal and phosphorus precipitated from Inositol tetraphosphate solution at pH's 4, 5 and 6. P/metal in Metal pH % metal % phosphorus precipitate Ca (as CaC12) 21.92 16.98 1.00 27.16 21.05 1.00 46.42 35.98 1.00 Cu (as CuSO4) 35.84 25.34 1.48 53.24 25.97 1.00 99.06 11.64 @ Fe (as F8804) 57.66 32.10 1.00 75.10 27.75 0.66 85.18 14.24 @ Zn (as ZnSO4) 12.62 5.98 1.00 61.33 24.43 0.84 63.32 20.54 @ @ These precipitates contain metal hydroxide and inositol tetraphosphate-metal complex. 74 Inositol pentaphosphate-metal complexes: The IDs-metal complexes were precipitated at all pH's (Table 12). The IP5-Ca complex upon analysis showed a phosphorus to calcium atom ratio of 1 : 1 at pH 4 and 6 : 5 at pH's 5 and 6. The IP Cu and IP5-Zn precipitates both displayed the phosphorus 5 to metal atom ratio of 1 : 1 at pH's 4 and 5. The IP -Fe 5 precipitates showed phosphorus to iron atom ratios of 1 : 1 at pH 4, and 1 : 2 at pH 5. Increasing the pH resulted in an increase in the percent of metal precipitated. The influence was more pronounced in calcium, copper and zinc precipitation than in iron precipitation. Calcium showed an increase in the atom ratio of P : Ca from 1 : 1 to 6 : 5 with an increase in the pH from 4 to 5 or 6. Increasing the pH from 4 to 5 did not change the atom ratio of both P : Cu and P : Zn from 1 : 1. For a similar increase in pH, the IP5-Fe precipitate showed a decrease in the P : Fe atom ratio from 1 : 1 to 1 : 2. Inositol hexaphosphate-metal complexes: Insoluble IP6-metal complexes were formed at all of the pH's studied (Table 13). The IP5Ca precipitate showed a phosphorus to calcium atom ratio of 6 : 5 at pH's 4, 5 and 6. Similar observations reported by Evans and Pierce (1981). Although a hexa-calcium phytate salt has been reported by Hay (1942), in a similar Table 12.--Percentages of metal and phosphorus precipitated from Inositol pentaphosphate solution at pH's 4, 5 P/metal in Metal % metal % phosphorus precipitate Ca (as CaC12) 26.46 20.51 1.00 47.76 44.06 1.19 53.11 48.89 1.19 Cu (as CuSO4) 52.55 25.64 1.00 78.06 38.08 1.00 98.90 10.15 @ Fe (as FeSO4) 63.76 35.39 1.00 68.76 19.08 0.50 97.70 29.28 @ Zn (as ZnSO4) 48.01 22.76 1.00 62.64 29.70 1.00 70.03 7.95 @ @ These precipitates contain metal hydroxide and inositol pentaphosphate-metal complex. 76 Table 13.--Percentages of metal and phosphorus precipitated from Inositol hexaphosphate solution at pH's 4, 5 and 6. P/metal in Metal pH % metal % phosphorus precipitate Ca (as CaC12) 28.10 25.77 1.18 31.46 29.16 1.19 39.44 36.82 1.19 Cu (as CuSO4) 78.94 38.51 1.00 92.64 45.18 1.00 99.90 21.22 @ Fe (as FeSO4) 75.16 41.72 1.00 75.44 41.87 1.00 82.10 33.39 @ Zn (as ZnSO4) 43.08 20.43 1.00 71.96 17.06 0.50 77.96 31.96 @ @ These precipitates contain metal hydroxide and inositol hexaphosphate-metal complex. 77 study Hoff-Jorgensen (1944) reported only the penta-calcium phytate. The IP6-Cu and IP6-Fe precipitate observed a phosphorus to metal ratio of 1 : 1 at pH's 4 and 5. The IPG-Zn complex showed a P : Zn atom ratio of 1 : 1 at pH 4 and 1 : 2 at pH 5. As the pH increased there was an increase in the percent of metal precipitated. The influence was more pronounced in copper and zinc. The calcium did not show any change in the atom ratio of P : Ca with an increase in pH, and was 6 : 5; Similar atom ratios were observed by Evans and Pierce (1981) over a pH range of 5 to 6. Increasing the pH from 4 to 5 did not change the atom ratio of P : Cu and P : Fe, which stayed at 1 : 1. Evans and Pierce (1982) showed phosphorus to copper atom ratio of 6 : 5 at pH 6, but copper starts forming hydroxides at pH 6 (Britton, 1925), thus the differences in the atom ratio may be due to hydroxide formation. The IP -Zn 6 precipitate observed an decrease in the atom ratio of P : Zn from 1 : 1 to 1 : 2 with a pH increase from 4 to 5. Phosphoric acid-metal complexes: The phosphoric acid-metal complexes were observed at all pH's studied except at pH 4 with zinc (Table 14). The H3PO4-Ca precipitate showed a phosphorus to calcium atom ratio of 1 : 1 at pH's 4, 5 and 6. The H3PO4-Cu complex analysis showed a phosphorus to copper atom ratio of 3 : 1 at pH 4 and 1 : 1 at pH 5. 78 Table 14.--Percentages of metal and phosphorus precipitated from phosphoric acid solution at pH's 4, 5 and 6. P/metal in Metal pH % metal % phosphorus precipitate Ca (as CaC12) 4 29.12 22.57 1.00 5 32.96 25.54 1.00 6 34.91 27.05 1.00 Cu (as CuSO4) 9.04 13.23 2.98 59.02 28.78 1.00 99.71 6.78 @ Fe (as FeSO4) 47.12 19.80 0.75 72.66 20.16 0.50 79.98 10.01 @ Zn (as ZnSO4) - - - 8.38 1.98 0.50 43.38 11.08 @ @ These precipitates contain metal hydroxide and phosphoric acid-metal complex. No precipitate formed. 79 The H3PO4-Fe precipitate observed a phosphorus to iron atom ratio of 3 : 4 at pH 4 and 1 : 2 at pH 5. The H3PO4- Zn complex isolated at pH 5 showed a atom ratio of 1 : 2. As the pH increased there was an increase in the percent of metal precipitated. The influence of pH was larger in copper and iron precipitation than in calcium and zinc precipitation. Increasing the pH from 4 to 6 did not change the atom ratio of the calcium precipitate, which stayed at 1 : 1. The H3PO4-Cu precipitate showed a decrease in the atom ratio of phosphorus to copper from 3 : 1 to 1 : 1 with an increase in pH from 4 to 5. The H3PO4-Fe precipitate also showed a decrease in the atom ratio of P : Fe, which changed from 1 : 1.32 to 1 : 2 with pH increase from 4 to 5. From the inositol phosphate-metal complex ratios, the possible structures that could be written are given in pages 80 to 83. 80 POSSIBLE STRUCTURES OF INOSITOL PHOSPHATE-METAL COMPLEXES INOSITOL MONOPHOSPHATE-METAL COMPLEX M I 0 O 119 (1.. (1:1)0M (1:2) MD INOSITOL DIPHOSPHATE-METAL COMPLEX o’M‘o \P/ p 6 P I 01:0 I (2 110'” M M : M I \ (1:1) 0: :0 0 p (1:2) 81 INOSITOL TRIPHOSPHATE-METAL COMPLEX +M M!- Q s5 , P (1:2) .P. \ (1,1) . .1 3. P‘ P‘ ' I \ 69 49 00 M M iaaMw (1:1) P-O-M-O-P (2:1) 9 B E B Os M I0 0 \M’0 INOSITOL TETRAPHOSPHATE-METAL COMPLEX M. ,M 6,9 0 ,b M 19 d 0 \Pl (1:1) +m .+ +8 MI 0,0 ,9 P—o-M-o-P P-O-M-O-P 3‘3: ‘ 6% t“ M+ +M M+ (1:1.5) 82 INOSITOL PENTAPHOSPHATE-METAL COMPLEX M M +M M++M M+ qu 6| [b +M+ Qprb 0\ b P M P (2,6 p P P on) or ’P‘ p\ M 'Pio ,P\ M+M+ P (1,9 A n+3 3+ ( ' 1) (1:2) INOSITOL HEXAPHOSPHATE-METAL COMPLEX 'Mb 'fib ,M\ M M P' b 0,9 0 0 . ‘P 1f 0.33 ,M. P va P ,P. P .Ji +M M+ +M M+ P 0MD ‘51? 0,9 on (1.0 6.30 6130 (6,5) M}! +M1> P M M . O\ (131) 83 PHOSPHORIC ACID-METAL COMPLEX CONCLUSIONS The yeast phytase had an optimum pH of 4.6 and optimum temperature of 45°C with acetate buffer and phytic acid as substrate. The Michaelis constant with phytate as substrate was 0.21 mM. The phytase could be separated from an asso- ciated phosphatase by DEAE-cellulose chromatography. The purified phytase shows a broad specificity being able to hydrolyze a number of phosphomonoesters besides phytic acid and other inositol phosphates and can be characterized as a nonspecific phosphomonoesterase with phytase and potent pyrophosphatase activity. This enzyme is inhibited by high concentrations of phytic acid. The enzyme activity is increased by'1 mM of Fe2+ and decreased by chelating agents. Inositol hexa-, penta-, tetra-, tri-, di-, and mono- phosphates along with inorganic phosphate were present in both whole wheat flour (WWF) and wheat flour (WF). IP6 was the dominant inositol phosphate in unfermented doughs prepared with either WWF of WF, although the WWF dough contained more than twice the amount of IP6 present in WF dough. The remaining inositol phosphates collectively contained almost as much P as IP6 in WWF dough, and 1.7 times as much as in WF doughs. Fermentation reduced the phytate content with the fastest decrease occurring during 84 85 the first 30 min. The rate of phytate degradation was greater in WWF dough than in WF dough. The content of intermediate inositol phosphates fluctuated with fermen- tation time and it was only after considering their phos- phorus content that an overall phosphorus balance could be achieved. The 70%, 80%, 90% and 100%-extraction flours obtained from soft red winter wheat flours contained all of the six inositol phosphates, plus inorganic phosphate. The amounts of Pi’ IP6 and total phosphorus increased with increasing extraction rate. The percent of IP6 phosphorus was 27%, 33%, 38% and 52% of all phosphates in the 70%- extraction, 80%-extraction, 90%-extraction, and 100%- extraction wheat flours, respectively. Dough fermentation for 2 hours reduced the phytate contents by about 70% in both 70%—extraction bread (E870) and 80%-extraction breads (E880), 67% in 90%-extraction bread (E890) but only 58% in 100%-extraction bread (E8100). As the phytate content decreased there was a concurrent increase in orthophosphate. After 2 hours of fermentation, the phytic acid contents in bread was already reduced to 7.9% to, 8.6% , 12.2% and 21.7% of total phosphate in E870, E880, E890 and E8100 respectively. A phosphate balance could be achieved only after taking intermediate inositol phosphates into account. Two commercial breads were also analysed for the entire spectrum of phosphates. The white bread contained 86 more Pi’ 1P6, and total phosphorus than expected, assuming a 70-75%-extraction. The excessive amount of Pi and total phosphorus were due to added phosphorus. The high amounts of phytic acid may be due to the inhibition of phytase by the added calcium. All six wheat inositol phosphates and phosphoric acid showed the ability to precipitate the minerals Ca, Cu, Fe and Zn at pH levels 4, 5 and 6, except IP1 and phosphoric acid, which did not precipitate zinc at pH 4. Calcium formed a simple atom ratio of 1 : 1 with all the inositol phosphates with following exceptions: with IP at pH 5, 1 IP at pH's 5 and 6, and IP6 at pH's 4, 5, and 6. In these 5 the ratio was 6 : 5. Copper showed a decrease in the atom ratio of P : Cu with an increase in pH from 4 to 5: 1P1, 3 : 1 to 1 : 1; IP and IP 1.5 : 1 2 3’ 4’ to 1 : 1; phosphoric acid, 3 : 1 to 1 : 1. Both IP5 and 2 : 1 to 1 : 1; IP IP6 did not show any changes in the atom ratio of 1 : 1, with an increase in pH. Thus copper formed a simple atom ratio of 1 : 1 at pH 5 with all inositol phosphates and phosphoric acid. Iron displayed a decrease in the atom ratio of P : Fe with an increase in pH from 4 to 5: 1P1, 1 : 2 to 1 : 3; IP and IP 1 : 1 to 1 : 2; IP and IP 2 5’ 3 4’ 1 : 1 to 1 : 1.5; phosphoric acid, 1 : 1.33 to 1 : 2. No change in atom ratio of 1 : 1 was observed in IP6-Fe complex for a similar rise in pH. Zinc showed an increase in molar ratio of 1 : 2 to 1 : 1 with an increase in pH 87 from 4 to 5 in both IP2 and 1P3. For a similar change in pH there was a decrease in atom ratios: IP4, 1 : 1 to 1 : 1.19 and 1P6, 1 : 1 to 1 : 2; while IP5 did not show any change in atom ratio of 1 : 1. LI TERATURE CI TED 88 BIBLIOGRAPHY AACC. 1962. "Approved Methods" 7th ed. American Association of Cereal Chemists, St. Paul, Minn. Agranoff, 8. W., Bradley, and R. 0. Brady. 1958. The enzymat- ic synthesis of inositol phosphatide. J. Biol. Chem. 233:1077. Allen, R. J. L. 1940. The estimation of phosphorus. Biochem .2; 34:858. Anderson, R. J. 1914. Concerning the organic phosphoric acid of cottonseed meal. J. Biol. Chem. 17:141. Anderson, R. J. 1914c. Concerning phytin in corn. J. Biol. Chem. 17:165. Asada, K. and Z. Kasai. 1959. Formation of phytin and its role in the ripening process of rice plant. Mem. Res. Inst. Food Sci., Kyoto Univ. 18:32. Asada, K. and Z. Kasai. 1962. Formation of myo-inositol and phytin in ripening rice grains. Plant Cell Physiol. 3:397. Asada, K., K. Tanaka, and Z, Kasai. 1968. Phosphorylation of myo-inositol in riepning grains of rice and wheat. Incorporation of phosphate-32p and myo-inositol-SH into myo-inositol phosphates. Plant Cell Physiol. 9:185. Ashton, W. M. and P. C. Williams. 1958. The phosphorus com- pounds oats. I. The content of phytate phosphorus. J. Sci. Food Agric. 9:505. Association of Official Analytical Chemists. 1975. Official methods of analysis. Washington, D. C. Averill, H. P. and C. G. King. 1926. The phytin content of foodstuffs. J. Am. Chem. Soc. 48:724. Barre, R., Courtois, J. E and Wormser, G. 1956. Action de quelques cations divalents sur diverses preparations glycerophosphatasiques et phytasiques du son de ble. Bull. Soc. Chim. Biol. 38:387 89 90 Barre, R. 1956. Influence de l'acide phytique sur 'la digestion pepsique de differentes protein. Ann. Pharm. Ea. 14:182. Belavady, B. and Banerjee, S. 1953. Studies on the effect of germination on the phosphorus values of some common Indian pulses. Food Res. 18:223. Britton, H. T. S. 1925. Electrometirc studies of the precip- itation of hydroxides. J. Chem. Soc. 127:2110. Biswas, S. and B. 8. Biswas. 1965. Enzymatic synthesis of guanosine diphosphate. Biochim. Biophys. Acta. 108:710. Bitar, K. and J. G. Reinhold. 1972. Phytase and alkaline phosphatase activities in intestinal mucosa of rat, chicken, calf and man. Biochim. Biophys. Acta. 268:442. Chang, C. W. 1967. Study of phytase and flouride effects in germinating corn seeds. Cereal Chem. 44:129. Common, R. H. 1939. Phytic acid and mineral metabolism in poultry. Nature, 143:379. Cosgrove, D. J. 1966. The chemistry and biochemistry of inositol polyphosphates. Rev. Pure Appl. Chem. 16:209 Courtois, J. 1947a. Recherches sur la phytase. IIlrEssais de separation de l'activite glycerophosphatasique et l'activie phytasique du son de ble. Biochim. Biophys. Acta. 1:270. Courtois, J. 1947b. Recherches sur la phytase. V.-Etude preliminaire de l'hydrolyse des inositohexaphosphates et glycerophosphates par les graines de Moutarde blanche. Bull. Soc. Chim. biol., Paris, 29:944. Courtois, J. and Joseph, G. 1947. Recherches sur la phytase. VI-Action de diverses preparations phosphatasiques sur quelques esters phosphoriques de l'inositol. Bull. Soc. Chim. biol., Paris, 29:951. Courtois, J. 1948. Recherches sur la phytase. VII.—Action de preparations phosphatasiques purifiees de moutarde- blanche sur divers esters phosphoriques. Bull. Soc. Chim. biol., Paris, 30:37. Courtois, J. and C. Perez. 1948a. Recherches sur la phytase. VIII. Teneur de inositophosphates et activite phytasi- que de diverses graines. Bull. Soc. Chim. Biol. 30:195. 91 Courtois, J. and C, Perez. 1948b. Recherches sur la phytase. XI.-Exxais en vue d obtenir des matieres premieres dont l activite phytasique initiate a ete articiellment accrue. Bull. Soc. Chim. Biol. 30:631 Courtois, J. and C. Perez. 1949. Presence dune phytase acide dans les feces humaines. Bull. Soc. Chim. Biol. 31:1373. Courtois, J. 1951. Les esters phosphorique l'inositol. Bull. Soc. Chim. biol., Paris, 33:1075. Davis, N. T. and R. Nightingale. 1975. The effect of phytate on intestinal absorption and secretion of zinc and whole body retention of zinc, copper, iron and manganese in rats. Br. J. Nutr. 34:243. Davis, P. N., L. C. Norris, and F. H. Kratzer. 1962. Inter- ference of soybean proteins with the utilization of trace minerals. J. Nutr. 77:217. de Boland, A. R., G. B. Garner, and B. L. 0 Dell. 1975. Identification and properties of "phytate" in cereal grains and oilseed products. J. Agric. Food Chem. 23:1186. de Lange, D. J., C. P. Joubert, and S. F. M. du Preez. 1961. The determination of phytic acid and factors which influence its hydrolysis in bread. Proc. Nutr. Soc. South Afric. 2:69. DeTurk, E. E., J. R. Holbert, and 8. W. Hawk. 1933. Chemical transformations of phosphorus in the growing corn plant, with results on two first-generation crosses. J. Agric. figs. (Washington, D.C) 46:121. Dixon, M. 1953. A nomogram for ammonium sulfate solutions. Biochem. J. 54:457. Earle, F. R. and R. T. Milner. 1938. The occurrence of phosphorus in soybeans. Oil Soap. 15:41. Ellis, L. C. and A. D. Tillman. 1961. Utilization of phytin phosphorus in wheat bran by sheep. J. Animal Sci. 20:606. Erdman, J. W. 1979. Oilseed phytates: nutritional implica- tion. J. Am. Oil Chemists Soc. 56:736. 92 Ergle, D. R. and G. Guinn. 1959. Phosphorus compounds of the cotton embryo's and their changes during germina- tion. Plant Physiol. 34:476. Evans, W. V. and A. G. Pierce. 1981. Calcium-phytate complex formation studies. J. Am. Oil Chem. 58:850. Fleury, P. and J. Courtois. 1947. Recherches sur la ; phytase. II.-Cinetiques comparees de l'hydrolyse du glycerophosphate et de l'inositohexaphosphate par le son de ble. Biochim. Biophys. Acta. 1:256. Forbes, R. M. and H. M. Parker. 1977. Biological availa- bility of zinc in and as influenced by whole fat soy flour in rat diets. Nutr. Repts. Intern. 15:681. Glass, R. L. and W. F. Geddes. 1959. Grain storage studies. XXVII.-The inorganic phosphorus content of deteriorating wheat. Cereal Chem. 36:186 Gibbins, L. N. and F. W. Norris. 1963. Phytase and acid phosphatase in the Dwarf bean, Phaseolus vulgaris. Biochem. J. 86:67. Hall, J. R. and T. K. Hodges. 1966. Phosphorus metabolism of germinating oat seeds. Plant Physiol. 41:1459. Harland, B. F. and J. Harland. 1980. Fermentation reduc- tion of phytate in rye, white and whole wheat breads. Cereal Chem. 57:226. Hay, J. G. 1942. The distribution of phytic acid in wheat and a preliminary study of some of the calcium salts of this acid. Cereal Chem. 19:326. Hoff-Jorgensen, E. K. 1944. Investigations on the solubility of calcium phytate. K. Dan. Vidensk. Selsk. Math. Fysisk. Medd. 21:NR 7. Jennings, A. C. and K. K. Morton. 1963b. Changes in nucleic acids and other phosphorus containing com- pounds in developing wheat grains. A g. J. Biol. Sci. 16:332. Johnson, L. F. and M. E. Tate. 1969. Structure of phytic acid. Can. J. Chem. 47:63. ' Kalckar, H. M. 1947. Differential spectrophotometry of purine compunds by means of specific enzymes. II.- Studies of the enzymes of purine metabolism. J. Biol. Chem. 167:461. 93 Kennedy, 8. M. and M. Schelstraete. 1975. Chemical, physical and nutritional properties of high protein flours and residual kernal from the over milling of uncoated milled rice. III. Iron, calcium, Magnesium, Phosphorus Sodium, Potassium, and Phytic acid. Cereal Chem. 52:173. Knorr, D., T. Watkins, and B. Carlson. 1981. Enzymatic reduction of phytate in whole wheat breads. J. Food. Sci. 46:1866. Likiski, H. J. A. and F. M. Forbes. 1965. IV. Effects of calcium and phytic acid on the utilization of dietary zinc. J. Nutr. 85:230. Lineweaver, H. and D. Burk. 1934. The determination of enzyme dissociation constants. J. Am. Chem. Soc. 56:658. Lolas, G. M. and P. Markakis. 1975. Phytic acid and other phosphorus compounds of beans. J. Agric. Food Chem. 23:13. Lolas, G. M., N. Palamidis. and P. Markakis. 1976. The phytic acidtotal phosphorus relationship in barley, oats, soybeans, and wheat. Cereal Chem. 53:867. Makower, R. U. 1969. Changes in phytic acid and acid- soluble phosphorus in maturing pinto beans. J. Sci. Fd. Agric. 20:82. Mandal, N. C. and B. B. Biswas. 1970. Metabolism of ino- sitol phosphates. I-Phytase synthesis during germina- tion in cotyledons of mung beans (Phaseolus aureus). Plant Physiol. 45: 4. Mandal, N. C. Burman, S. and B. B. Biswas. 1972. Isolation, purification and characterization of phytase from germinating mung beans. Phytochemistry. 11:495. Mayer, A. M. 1958. The breakdown of phytin and phytase activity in germinating lettuce seeds. Enzymologia. 19:1. McCance, R. A. and Eq M. Widdowson. 1944. Activity of the phytase in different cereals and its resistance to dry heat. Nature (London). 153:650. Mellanby, E. 1944. Phytic acid and phytase in cereals. Nature (London). 154:394. 94 Michel-Durand, E. 1939. Le phosphore des vegetaux (Presses Univ-de France, Paris). Mihailovic, M. L., M. Antic, and D. Hadzijev. 1965. Chem- ical investigation of wheat. 8 -Dynamics of various forms of phosphorus in wheat during its ontogenesis. The extent and mechanism of phytic acid decomposition in germinating wheat grain. Plant and Soil. 23:117. Momcilovic, B. and B. G. Shah. 1976b. Bioavailability of zinc in infant foods. Nutr. Rep.#Int. 14:717. Morris, E. R. and R. Ellis. 1976. Isolation of monoferric phytate from wheat bran and its biological value as an iron source to the rat. J. Nutr. 106:753. Morton, R. K. and J. K. Raison. 1963. A complex ontrace- llular unit for incorporation of amino acids into storage protein utilizing adenosine triphosphate gen- erated from phytate from phytate. Nature (London). 200:429. Myers, D. V. and G. A. Iacobucci. 1974. Binding of phytic acid to glycinin. Cereal Science Today. 19:401. Nagai, Y. and S. Funahashi. 1962. Phytase (myoinositol hexaphosphate phosphohydrolase) from wheat bran. Part I.-Purification and substrate specificity. Agr. Biol. Chem. 26:794. Oberleas, D., M. E. Muhrer. and B. L. 0 Dell. 1962. Effects of phytic acid on zinc availability and parakeratosis in swine. J. Anim. Sci. 21:57. Oberleas, D., M. E. Muhrer. and B. L. 0 Dell. 1966. Diet- ary metal-complexing agents and zinc availability in the rat. J. Nutr. 90:56. O'Dell, B. L., A. R. de Boland, S. R. Koirtyohann. 1972. Distribution of phytate and nutritionally important elements among the morphological components of cereal grains. J. Agr. Food Chem. 20:718. Okubo, K.,+ . A. Iacobucci, and D. V. Myers. 1974. Effects of Ca on phytate binding to glycinin. Cereal Science Today. 19:401. ‘ Okubo. K., D. V. Myers, and G. A. Iacobucci. 1976. Binding -of phytic acid to glycinin. Cereal Chem. 53:513. 95 Peers, F. G. 1953. The phytase of wheat. Biochem. J. 53:102. Pfeffer, E. 1872. Pringsheim jahrbucker fur wiss. Bot. 8:429, 475, cited by Courtois, J. 1951. Les esters phosphoriques de linositol. Bull. Soc. Chim. 8101., Paris. 33:1075. Pileggi, V. J. 1959. Distribution of phytase in the rat. Arch. Biochem. Biophys. 80:1 Pomaranz, Y. 1973. Structure and mineral composition of cereal aleurone cells as shown by scanning electron microscopy. Cereal Chem. 50:353. Prasad, A. S., A. Miale., Z. Farid., H. H. Sanstead., A. R. Schubert, and W. J. Darby. 1963. Biochemical Studies on Dwarfism, Hypogonadism and Anemia. Arch. Internal. Med. 111:407. Preece, I. A. and H. J. Grav. 1962. Studies on phytin. 11.-Preliminary study of some barley phosphotases. J. Inst. Brew. 68:66. Preece, I. A., H. J. Grav, and A. T. Wadham. 1960. Studies on phytin. I.-The inositol phosphatases. J. Inst. Brew. 66:487. . Ranhotra, G. S. 1972. Hydrolysis during breadmaking of phytic acid in wheat protein concentrate. J. Food Sci. 37:12. Ranhotra, G. S. 1973. Factors affecting hydrolysis during breadmaking of phytic acid in wheat protein concen- trate. Cereal Chem. 50:353. Ranhotra, G. S., C. Lee, and J. A. Gelroth. 1978. BIO? availability zinc in soy-fortified wheat bread. Nutr. Rep. Int. 18:487. Ranhotra, G. S., R. J. Loewe, and L. V. Puyat. 1974. Phytic acid in soy and its hydrolysis during bread- making. J. Food Sci. 39:1023. Raun, A., E. Chang, and W. Burroughs. 1956. Phytate phos- phorus hydrolysis and availability to rumen microor- ganisims. J. Agric. Food Chem. 4:869. Reinhold, J. G. 1971. High phytate content of rural iranian bread: a possible cause of human zinc deficiency. Am. J. Clin. Nutr. 24:1204. 96 Reinhold, J. G. 1972. Phytate concentrations of leavened and unleavened iranian breads. J. Food Nutr. 1:187. Reinhold, J. G., F. Ismail-Beigi, and B. Faradji. 1975. Fibre vs phytate as determinant of the availability f30f calcium, zinc and iron of breadstuffs.v Nutr. Rept. Int. 12:75. Reinhold, J. G., H. Hedayati., A. Lahimgarzadeh, and K. Nasr. 1973. Zinc, calcium, phosphorus, and nitrogen balances of iranian villagers following a change from phytate-rich to phytate poor diets. Ecol. Food Nutr. 2:157. Reinhold, J. G., A. Lahimgarzadeh., N. Khosrow, and H. Hedayati. 1973. Effects of purified phytate and phytate-rich bread upon metabolism of zinc, calcium, phosphorus, and nitrogen in man. Lancet. 1:283. Rush, R. M. and J. H. Yoe. 1954. Colorimetric determina- tion of zinc and copper with 2-carboxy-2-hydroxy-5- sulfoformazyl benzine. 'Anal Chem. 26:1345. Saio, K. 1964. The change in inositol phosphates during the ripening of rice grains. Plant and Cell physiol. 5:393. Saio, K., E. Koyama, and T. Watanabe. 1967. Protein- calcium-phytic acid relationships in soybeans. Part I.-Effects of calcium and phosphorus on solubility characteristics of soybean meal protein. Agric. Biol. Chem. 31:1195. Saio, K., E. Koyama, and T. Watanabe. 1968. Protein- calcium-phytic acid relationships in soybean. Part 11.-Effects of phytic acid on combination of calcium with soybean meal protein. Agric. Biol. Chem. 32:448. Samotus, B. 1965. Role of phytic acid in tuber. Nature (London) 206:1372. Sloane-Stanley, G. H. 1961. Phytase, in Biochemists Hand- book. Ed. C. Long. (E. and F. N. Spon, London). Smith, A. K, and J. J. Rackis. 1957. Phytin elimination in soybean protein isolation. J. Amer. Chem. Soc. 79:633. Sobolev, A. M, and M. A. Rodionova. 1966. Phytin synthesis by aleurone grains in ripening sunflower seeds. Sov. Plant Physiol. 13:958. 97 Suzuki, U., K. Yoshimura, and M. Takaishi. 1907. Ueber ein Enzym "Phytase" das "Anhydro-oxy-methylen diphos- phorsaure" spaltet. Tokyo Imperial Univ Coll. Agric. Bull. 7:503. Tangkongchitr, U., P. A. Sieb, and R. C. Hosney. 1981. Phytic acid. II. Its fate during breadmaking. Cereal Chem. 58:229. Tombs, M. P. 1967. Protein bodies of the soybean. Plant Physiol. 42:797. Turnbull, A., F. Cleton., C. A. Finch., L. Thompson, and J. Martin. 1962. Iron absorption. IV. The absorption of hemoglobin iron. J. Clin. Invest. 41:1897. Walker, K. A. 1974. Changes in phytic acid and phytase during early development of Phaseolus vulgaris. Planta. 116:91. Wang, L. C. 1971. Effect of phytate in isoelectric_focusing of soybean whey proteins. Cereal Chem. 48:229.’ Warburg, O. and W. Christian. 1942. Isolierung und Kris- takisation des Garungsferments Enolase. Biochem. Z. 310:384. Watanabe, F. S. and S. R. Olsen. 1965. Test of an ascorbic acid method for determining phosphorus in water and NaHCO; extracts from soil. Soil Sci. Soc. Am. Proc. 29:67 . ' Whitaker, J. R. 1972. Principles of enzymology for the Food Sciences. Marcel Dekker, Inc., N. Y. Williams, S. G. 1970. The role of phytic acid in the wheat grain. Plant Physiol. 45:376. Winterstein, E. 1897. Ueber einen phosphorhaltigen pflan- zenbestandtheil welcher bei der spaltung liefert. Ber Dtsch. Chem. Ges. 30:2299. Wozenski, J, and M. Woodburn. 1975. Phytic acid (Myoino- sitol hexa phosphate) and phytase activity in flour cotton seed protein products. Cereal Chem. 52:665. APPENDIX 1 Table.--Recovery of phytic acid added to the control sample PA in Adged Total PA % Recovery Sample control PA PA found sample (mg) (mg) (mg) (mg) Control 12.05 - - —' - Control + 10 mg 12.05 5.24 17.57 16.57 95.84 NaPhy Control + 20 mg 12.05 10.47 22.52 21.37 94.89 NaPhy . a Average of three determinations. b PA = phytic acid + 52.37% of standard NaPhy. "lllllllliliilllllllillf