1A.}. lil\lu‘.n‘ll1...:.a lvl \.luc.. '0 tn! U 1 . . ‘II‘ $n| NI. 1 {I .7 73. ‘1....J.v‘,- ‘ ‘lIV\‘O.\l 1y . T ‘17 la, .A.\|‘v.fl¢lvl It 3dnrc.fi.u.fi.l. ... nay 6.0! 101’! (b. . tl“ li‘ifikl III-II. .‘ll I‘ll)\|."lrl\lllll I .11“ ‘ tn. Jhflfi‘fluhnul'l‘nlnufln . t > I (“I-I1 Jr- ‘ . I‘D I‘l ‘ l] llllllllllllllllllllllllllllllllllllllllllllll 3 1293 105790 Date 0-7639 --.uL-._._.A ._ ‘_.‘._,A_,_.i ‘ i I E 4, , " l I ’ 1 ‘ 11.,- :hi fi- 3..” .- t a r. 'f' -“‘A 47 91“- L. 1.. K." :..i' o . _. 10“ “‘YJMJL-‘KJ ‘~‘,' ”fit- 1!“ JV ilk ‘1' \l > 5 a? .‘1 " e. 2"“ °W 7‘73"?“ ' \ /' "———.v _ -...__.-_ . .-- -.‘_~ w- This is to certify that the thesis entitled PHYTIC ACID AND MINERAL PARTITIONING AND IRON BIOAVAILABILITY FROM AIR CLASSIFIED BEAN FLOUR FRACTIONS presented by Elaine Tecklenburg has been accepted towards fulfillment of the requirements for M ' S ‘ degree in FOOd SC I ence l 7 t r ajor profebé‘ Juli 11, 1983 MS U is an Affirmative Action/Equal Opportunity Institution MSU LIBRARIES .1“. vww" RETURNING MATERIALS: Place in book drop to remove this checkout from your record. FINES will. be charged if book is returned after the date stamped below. PHYTIC ACID AND MINERAL PARTITIONING AND IRON BIOAVAILABILITY FROM AIR CLASSIFIED BEAN FLOUR FRACTIONS By Elaine Tecklenburg A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Food Science and Human Nutrition 1983 ABSTRACT PHYTIC ACID AND MINERAL PARTITIONING AND IRON BIOAVAILABILITY FROM AIR CLASSIFIED BEAN FLOUR FRACTIONS By Elaine Tecklenburg Hull flours and air classified intermediate starch, high starch, and high protein navy, pinto, and black bean flours were analyzed for calcium, copper, iron, magnesium, sodium, phosphorus, and zinc by plasma emission spectroscopy and for phytic aCid by the method of Wheeler and Ferrel (1971). Phytic acid content ranged from 4.29 to 8.72 mg/g in the high starch flours to 23.74 to 30.22 mg/g in the protein flours. Partitioning of phosphorus, zinc, iron, potassium, and magnesium into the protein flours was also noted. Strong and significant correlation coefficients were obtained between phytic acid content and phosphorus, iron, zinc, magnesium, and potassium content, and between the content of these minerals and protein content. As measured by hemoglobin regeneration in anemic rats, iron bioavailability from navy hull, high starch, and protein flours did not appear to be influenced by endogenous phytic acid. ACKNOWLEDGMENTS Many individuals deserve recognition for the assistance they provided during the course of this study. Without the knowledge, talents, various technical skills, physical endurance, and/or emotional support they contributed, this research project might not have been accomplished. Thanks to all who made it possible: Dr. N. Chenoweth Dr. R. Herner Dr. M. Uebersax Dr. M. Zabik Larry Baner Beth Martin Ma Belle Margaret Schiebner Barb Campbell Peggy Spink Norm Hord Barb Nassink Katie Jubell Marce Weaver Earl Klugh Baby X Dr. Ku it TABLE LIST OF TABLES. . . . . . . . LIST OF FIGURES . . . . . . . INTRODUCTION. . . . . . . . . REVIEW OF LITERATURE. . . . . Dry Bean Composition . . Proteins. . . . . . Amino Acids . . . . Fats. . . . . . . . Carbohydrates . . . Ash and Minerals. . Vitamins. . . . . . Antinutritional Factors Phytic Acid . . . . . . Phytase . . . . . . . . OF CONTENTS Metallic Phytates. . . . . . . Nutritional Significance of Iro Other Factors Influencing Iron Absorptio n Phytate Iron Absorption, Transfer, and Storage. Feeding Studies Relating Iron Absorption to Phytic Acid Intake . . . . . . . MATERIALS AND METHODS O O O O O O O O O O O 0 Raw Material Procurement and Processing. Source............... Roasting and Dehulling. . . . . Grinding and Air Classification Sample Materials. . Proximate Analyses . Moisture. . . . . . Ash . . . . . . . . Protein . . . . . . Crude Fat . . . . . Dietary Fiber . . . Starch. . . . . . . Mineral Analysis . . . Equipment Handling. Sample Preparation. . . ICP Emission Spectromete r 111 n O O O O O O O O O O O O O O O 0 PAGE Phytic Acid Analysis . . . . . . . Feeding Study For Determining Iron Animals and Depletion Treatment . . . . . . . Experimental Diets and Treatments . . . . . . Repletion Parameters. . . . . Statistical Analyses of the Data . RESULTS AND DISCUSSION. 0 O O O O O O O Proximate Composition. Moisture. . . . . Ash . . . . . Protein . . . Fat . . . . . Dietary Fiber Starch. . . . Mineral Analysis . Calcium . . . Copper. . . Iron. . . . Magnesium . Phosphorus. Zinc. . . . Sodium. . . Potassium . . . Related Research Findings Materials Balance Analysis of Phytic Acid Content. . . . . . . . Materials Balance Analysis of Bioavailability Mineral Content e Phytic Acid Cont n Correlations Between Mineral Content, Phytic Acid Content, and Macroconstituents . . . . . Feeding Study for Determining Iron Rationale . . . . . . . . . . Repletion Data. . . . . . . . SUMMARY AND CONCLUSIONS . . . . . . . . REFERENCES. 0 O O 0 O O O O 0 O O O O 0 APPENDIX. 0 O O O O O I O O O O O O O 0 iv Bioavailability t LIST OF TABLES Table Page 1 Range in content of selected mineral elements of raw, mature Phaseolus vulgaris classes reported in the 1iterature0 O O O O O O O O O O O O O O O 0 O 7 2 Range in content of selected vitamins of raw, mature Phaseolus vulgaris classes reported in the literature 0 O O O O O O O O O O O O O O O O 0 O O O 11 3 Composition of the basal low iron diet . . . . . . . 45 4 Proximate composition of various navy bean flour fractions. 0 O O O O O O O O O O O O O O O O O O 0 O 49 5 Proximate composition of various pinto bean flour fraCti 0'15. 0 O O O O O O O O O O O O O O O I O O O O 49 6 Proximate composition of various black bean flour fraCtionSo O O O O O O O O O O O O O O O O O O O O O 50 7 Average content of eight minerals in various navy bean flour fractions . . . . . . . . . . . . . . . . 56 8 Average content of eight minerals in various pinto bean flour fractions 0 O O O O O O O O O O O O O O O 57 9 Average content of eight minerals in various black bean flour fractions . . . . . . . . . . . . . . . . 58 10 Calculated mineral content of original navy, pinto, and black beans based on materials balance information 0 O O O O O O O O O O O O O O O O O O O O 71 11 Average phytic acid content (mg/g) of various navy, pinto and black bean flour fractions . . . . . . . . 74 12 Calculated phytic acid content of original navy, pinto and black beans based on materials balance information I O O O O O O O O O O O O O O O O O O O O 77 13 Correlation coefficients between various macro and micro constituents of navy bean flour. . . . . . . . 79 Table 14 15 16 17 18 19 20 21 Correlation coefficients between various macro and micro constituents of pinto bean flour. . . . . . . . Correlation coefficients between various macro and micro constituents of black bean flour. . . . . . . . Repletion parameters of anemic rats fed basal low iron diet and test diets containing navy hull, starch II, and protein flour fractions for 14 days. . Iron content of test and standard diets determined in repeated analyses. . . Composition of standard diets estimated to contain 6, 12, and 24 ppm iron from ferrous sulfate . . . . . Composition of test diets and 24 ppm iron from navy Composition of test diets and 24 ppm iron from navy Composition of test diets and 24 ppm iron from navy vi estimated to contain 6, 12, hu1] flour. 0 O O O O O O O estimated to contain 6, 12, starch II flour . . . . . . estimated to contain 6, 12, protein flour . . . . . . . Page 80 81 86 87 99 100 101 102 Figure 10 11 12 13 14 15 LIST OF FIGURES Structure of phytic acid pr0posed by Anderson (1914). Flow diagram of flour processing scheme . . . . . . Percentage yield of flour fractions obtained from navy, pinto and black beans . . . . . . . . . . . . Proximate composition of various navy bean flour fraCtionS O O O O O O 0 O O O O O O O O O O O O O O Proximate composition of various pinto bean flour fraCtions O O O O O O O O O O O O O O O O O O O O O Proximate composition of various black bean flour fractions 0 O O O O O O O I I O O O O O I O O I O 0 Average calcium content of various navy, pinto and black bean flour fractions. . . . . . . . . . . . . Average copper content of various navy, pinto and black bean flour fractions. . . . . . . . . . . . . Average iron content of various navy, pinto and black bean flour fractions. 0 O C C O O O O O O O O O O 0 Average magnesium content of various navy, pinto and black bean flour fractions. . . . . . . . . . . . . Average phosphorus content of various navy, pinto and black bean flour fractions. . . . . . . . . . . . . Average zinc content of various navy, pinto and black bean flour fractions. . . . . . . . . . . . . . . . Average sodium content of various navy, pinto and black bean flour fractions. . . . . . . . . . . . . Average potassium content of various navy, pinto and black bean flour fractions. . . . . . . . . . . . . Average phytic acid content of various navy, pinto and black bean flour fractions. . . . . . . . . . . vii Page 16 35 37 51 52 53 60 61 63 64 65 67 68 69 75 INTRODUCTION Although legumes contain approximately twice as much protein as cereal grains, it is well recognized that they remain an underutilized food source for man, particularly in the United States. Several reasons for the low level of consumption have been proposed, including flatulence associated with legume consumption, the presence of antinutritional factors in unheated legumes, and especially, the long soaking and cooking times required to adequately soften legumes. Seeking alternative uses for dry beans in order to increase their utilization, researchers have experimented with flours prepared from different bean varieties in various baked and extruded products. Air classification has permitted the separation of dehulled whole bean flours into high protein and starch fractions which offer more desirable functional and sensory characteristics than the whole bean flours for making specific products. While the composition of whole beans, and therefore whole bean flours, has been established, less is known about the nutritional contribution made by various air classified and hull flour fractions. Specifically, little information exists regarding the distribution of nutritionally important elements and the heat stable antinutritional factor, phytic acid, in these flours. Phytic acid has the potential to bind mono and divalent cations, most notably zinc and iron, possibly decreasing their bioavailability. This study was undertaken to determine the quantity and partitioning pattern of eight important dietary minerals as well as phytic acid in each of five flour fractions of navy, pinto, and black beans. For each bean type, hull, intermediate starch, high starch, high protein, and dehulled whole flour fractions were produced. After obtaining data on mineral content, phytic acid content, and proximate compostion of each flour fraction of each bean type, it was possible to attempt to correlate the individual microconstituents with macroconstituents, thus providing an indication of with which macrocomponents the microcomponents partitioned. To demonstrate whether or not iron bioavailability was affected by any phytic acid- iron chelates which might have been present, hemoglobin regeneration in anemic rats fed diets containing three of the navy bean flour fractions (high starch, high protein, and hull) was assessed relative to rats fed standard diets containing ferrous sulfate. REVIEW OF LITERATURE The Phaseoleae subfamily of the Leguminosae contains 47 genera including Phaseolus with 150 Species. Despite this large number, only a select few of these species are actually cultivated. Food legumes belonging to the Phaseolus genus are often referred to by various locally recognized names including common, kidney, field, garden, or haricot beans. The best known and most widely cultivated species is Phaseolus vulgaris, to which the majority of legumes grown in America belong. Dry beans which may be classified into this group include pinto, black, small white or navy, red, yellow eye, and great northern (Deschamps, 1958). Dry Bean Composition Proteins Dry beans contain substantial quantities of several important macro and micronutrients. Legumes have a high protein content, averaging between 20 and 25% on a dry weight basis. The average protein content of legumes is twice that found in cereal crops on a per serving basis (Bressani, 1975). However this is only about half as much protein as is contained in a serving of lean meat (Charley, 1970). The predominant class of proteins present in seeds of Phaseolus are salt-soluble globulins of which three distinct proteins have been identified; phaseolin, phaselin, and conphaseolin (Bressani, 1975; Kay, 1979). Analysis of the seed coat or taste, which comprises between 6.6 and 9.2% of the total weight of the bean (Kay, 1979), shows that the hull has a crude protein content of approximately 6.0% (Tobin and Carpenter, 1978). Amino Acids Legumes as a group have been shown to contain relatively large amounts of lysine, making them an excellent choice to complement cereals in order to achieve complete amino acid patterns. Legumes are also better sources of isoleucine, leucine, phenylalanine, threonine, and valine than are cereals (Charley, 1970; Bressani, 1975; Tobin and Carpenter, 1978). Legumes, like cereals, tend to be somewhat deficient in methionine. The exact amino acid content of legumes depends on the species, varieties, localities, and management practices. The application of minor element fertilizers has been found to influence the amino acid composition of certain legume varieties. Studied separately, uptake of both zinc and sulfur by Pisum sativum caused increases in the methionine content of mature peas (Bressani, 1975). Many studies have been conducted to assess the quality of legume protein both with and without supplementation or complementation by various cereal grains, seeds, and nuts (Rockland and Radke, 1981; Sgarbieri et al., 1978; Baloorforooshan and Markakis, 1979; Kakade and Evans, 1965). Determining and improving the protein quality of legumes is considered particularly important in parts of Africa, Asia, and Latin America where cereals and tubers are major components of the diet (Sinha, 1977). In countries such as the United States, however, meat, eggs, and milk provide the majority of the protein in the diet such that the protein quality of legumes is of little importance except for those individuals following strict vegetarian diets. Fats Legumes of the Phaseolus vulgaris species have a low fat content, generally below 2.0%, with the majority (63.3%) of the fatty acids being unsaturated (Deschamps, 1958; Watt and Merrill, 1979). Carbohydrates The average carbohydrate content of Phaseolus seeds is approximately 60% of the dry weight of the bean (Kay, 1979). Of this 60%. starch is typically the carbohydrate present in greatest abundance, reported to account for about 35.2% of the dry weight. As for other carbohydrates, Kay (1979) reported the following percentages: pentosans, 8.4%; dextrins, 3.7%; cellulose, 3.1%; sugars, 1.6%, and galactans, 1.3%. Walker and Hymowitz (1972) found a considerably higher sugar content in the 28 varieties of Phaseolus vulgaris they analyzed, ranging from 4.4 to 9.2%. Of the total sugar present, on average, sucrose accounted for 46.4%; raffinose, 10.4%; and stachyose, 43.0%. Raffinose, stachyose, and verbascose are the three, four, and five unit oligosaccharides considered to be, at least in part, responsible for the flatulence associated with bean consumption (Tobin and Carpenter, 1978). Crude fiber values of less than 6.0% have been noted for the more common varieties of the genus Phaseolus (Deschamps, 1958; Tobin and 6 Carpenter, 1978; Kay, 1979). According to many nutritionists, however, crude fiber, which is defined as the residual insoluble organic matter after digestion for a set period of time first in acid and then in alkali, does not provide an accurate indication of the total amount of unavailable carbohydrate. As such, more recently the amount of dietary fiber of Phaseolus vulgaris, defined as the plant polysaccharides plus lignin which are resistant to hydrolysis by man's digestive enzymes, has been reported as 225 g/kg or 22.5% (Tobin and Carpenter, 1978). Ash and Minerals It is generally agreed that the total ash content of P. vulgaris falls between 3.5 and 4.1% (Fordham et al., 1975; Tbbin and Carpenter, 1978; Kay, 1979). The content of specific minerals in mature, raw legumes has been reported by several researchers in recent years, however most values have pertained to beans grown in countries other than the United States. Since the soil composition, growing conditions, and varieties of legumes grown in North America differ somewhat from those in other parts of the world, only findings for beans grown in the U.S. are discussed. The range in content of several minerals for various classes of raw P. vulgaris which have been reported in the literature appear in Table 1. All researchers except Walker and Hymowitz (1972) and Augustin et al. (1981) used atomic absorption (AA) spectrometry to measure the content of all minerals except phosphorus. Walker and Hymowitz (1972) employed emission spectrosc0py in their analyses, and both Augustin et al. (1981) and Meiners et al. (1976) measured phosphorus colorimetrically. Although Augustin et al. (1981) used AA .eoua—aco any: «coon :ou.xo: up; can .eu.—ou .ou:.n .90; .uupz3o .vo~»—aco use: meson nocv.g to; ecu .00.:3 .aua—a .»,uzm .vouxpaco cum: mecca aucc.a to; use .oac.a .gcogaco: yucca .asazo .vouapuzo can: mean; mace—g to; tag .ouc—a .x:.a .vus pp-Em .vuua—oco ago: «some ou=.n ecu .auev.g vac .»>o= .ccogucoc usage” .uw~»—~=a 0cm: memos oa.;: ppusm can .uoc ppuem .ouc.n .gc—a .aocv.a to; .a>~: .ecoguso: yucca .ausoacusu .xuapc— - 872: 8278»... 28-88 - - 3-3 .. $2-2: m.38: :25: 2. to: m~-~_ . coco—-co-~ ooomuoomw cwuu comuuocm— mm—uoo Nu-» cowuuoc—~ m.-m—. nacxbss: tee Lou—ox m~-_~ mm-~_ scan—tmomm oc_m-ce~n m~1c~ nos—uenm— mssnm. elk muouumam «Aosuuv .p- a0 «soc—u: 21mm - 8:723. - - - 8-2 - 88.82 «:8: 9:8 2. 6:33. - - co~m~1ommc_ oocmuomem ~m~-m~_ --uc~o~ mound a weenuonon «Ammo—v .—a as légvcou mo-m~ o—~-ce cams—-oe~n_ oesm-eoon c~-o_ ocm~-cce_ om-nn o_-m oo--co~ ~.umo~. .pu uo c—auaoac :N oz x a c: a: on :9 cu oucosouox Eng .wcauucwu._ egg :. vouconwc momma—u m_ccm~=> mspoomoza eczema .xcc we macusu—o paguc.s vogue—om mo acoucou :. once: .~ u—aa» 8 to measure calcium, magnesium, zinc, copper, and iron; sodium and potassium were measured by flame emission spectrometry. In general, the data reported by all these workers indicate that dry beans contain substantial amounts of calcium, iron, magnesium, phosphorus, and potassium. Overall, the mean values for each mineral determined by the various research teams are in close agreement except for sodium and manganese. In addition to high interlaboratory variability of manganese content, intralaboratory variability was also found to be high by both Fordham et al. (1975) and Augustin et al. (1981). Although the beans analyzed by Fordham and coworkers (1975) were purchased in a local Kentucky market and the precise growing regions were not known, these researchers pointed out that the concentration of manganese in vegetables has been shown to vary by as much as 14 fold depending on the locations from which the samples were obtained. This may in part explain the large range of manganese values appearing in the literature. Augustin et al. (1981) noted large variability both within and between bean types for sodium content and attributed it to the inherently low sodium content of beans. However, Meiners et al. (1976) found lower sodium values with much smaller variability in their work, perhaps reflecting a difference due to the method of analysis. Since sodium and other elements such as iron, zinc, and calcium must be considered ubiquitous, errors which could possibly result from contamination should not be overlooked when considering sources of variability. In terms of other minerals, Meiners et al. (1976) found significant differences among the four .3. vulgaris bean types purchased in a local Virginia market for all minerals except phosphorus, however no one bean was consistently higher or lower in any one mineral than the other three types. Similarly, Augustin et al. (1981), in studying nine classes of raw 3. vulgaris grown in six locations (CA, CO, ID, NE, MI, and ND) found low variability for both phosphorus and magnesium, whereas higher variability was noted for all other minerals analyzed. These workers also found that while potassium showed low variability both between and within bean classes when grown in the same location, differences within classes grown in different areas were much larger. Perhaps the somewhat high potassium content reported by Koehler and Burke (1981) for beans grown in the state of Washington may also be due to a location or soil difference. The zinc content of these beans was also slightly higher than the values reported by the other workers. Whereas Meiners et al. (1976) noted the least variability for zinc content, Augustin et al. (1981) found the overall variability of this element to be high. It may be hypothesized that the variation in zinc content and other minerals analyzed is likely the result of soil or location differences, stage of maturity at harvest, and the analytical technique employed in the laboratory. Walker and Hymowitz (1972) found significant negative correlations between fat content and zinc, iron, and calcium contents of 28 varieties of P. vulgaris studied, perhaps suggesting that these elements are not associated with the lipid material present in beans. Significant correlation coefficients were also produced between raffinose content and phosphorus and potassium contents, and between stachyose and protein content. Although these correlation 10 coefficients were statistically significant, all were less than 0.60, suggesting that the relationships between the organic components and the inorganic elements of beans are not directly proportional. Vitamins There is no evidence in the literature which indicates that dry beans contain appreciable amounts of fat soluble vitamins. The quantity of vitamin A reported for P. vulgaris classes is less than 30 International Units per 100 grams of raw beans (Watt and Merrill, 1963; Kay, 1979). The tocopherol content of four varieties of .5. vulgaris seed averaged only 1.2 mg/100 g (Fordham et al., 1975). In terms of water-soluble vitamins, dry beans have been found to contain high levels of thiamin, riboflavin, niacin, and folic acid, but they contain very little (less than 5 mg/100 g) ascorbic acid (Watt and Merrill, 1963; Fordham et al., 1975; Tbbin and Carpenter, 1978). A summary of the range in content of several water-soluble vitamins contained in raw, mature P, vulgaris seeds is presented in Table 2. The data are in close agreement for the majority of these nutrients. Average thiamin content showed the highest interlaboratory variability of all the vitamins studied, with values ranging from 0.64 to 1.07 mg/100 g. Augustin et al. (1981) suggested that the high variability in values reported in their laboratory for this nutrient might be the result of different growing locations and possibly different sampling times following harvest. Values reported for various vitamins analyzed were expected to decrease as the amount of time between harvest and analysis increased. In assessing both the vitamin and mineral content of dry beans it is important to recognize that soaking and cooking by conventional 11 .uuszucm use: meson cmovxmz vac use .ouvpou .oucwa .uog .muvgze m N .vo~»pm=m may: xpco mason oucva .cmwxpmca use; memos oucva new .xocupx was .x>mc .ccocuco: “coco .um~apucm any: magma ou.g3 pmem new .umc ppaEm .oucpn .xcya .accurx no; .A>oc .ccmzugoc pumcm .xgcmacmco .xanmH - - . oe.~-o~cm -.o-o~.o em.o-fim.c eamamfiv __.Lcaz was “an: o~.o m~.o o.H - om.o mAccmHV .Fa up u=a_xuom - - .e_.m-mo.~ m~.o-¢~.o mm.H-wm.o NAmeH. .Pa um Engaged mm.o-cm.o mo.o-mfi.o H~.m-mm.o He.o-HH.o NM.H-~m.o “Aflmmfiv ._a pm =.am=m=< mcvxouvcxa c—u< u_—ou :Fuowz cp>mpwoawm cram—ch mocccmwwm m sea son as .mcauogmuvp ecu c? vmycoamc mommmpu m2cumps> mapoomosa assume .zoc mo mcwsouv> venom—mm we ucwucou :F magma .N «Fame 12 methods will result in losses of the water soluble forms of these nutrients. The extent of losses due to heat treatment and leaching will depend on the specific nutrient, the length of soaking, the amount of water used, and the severity of the cooking process (Miller et al., 1973). In the study conducted by Augustin et al. (1981), retention values exceeded 70% for water-soluble viatamins and 80% for all minerals except sodium, for which retention was in the 40% range. In the case of dry roasted bean flours being incorporated into baked products, no soaking takes place, thus the losses due to leaching would presumably be eliminated. Antinutritional Factors Although some vitamins and minerals may be lost by soaking and cooking, these processes have been found to result in the inactivation or removal of certain antinutritional factors present in P, vulgaris. Bressani (1975) categorized the toxic substances present in legumes into the following seven groups: trypsin inhibitors, hemagglutinins or lectins, goitrogenic factors, cyanogenic glucosides, lathyric factors, compounds that cause favism, and other factors about which much less information is known. Of these factors, trypsin inhibitors and hemagglutinins have been considered primarily responsible for causing the growth retardation observed in laboratory animals fed raw 3. vulgaris (Honavar et al., 1962; Liener, 1962; Kakade and Evans, 1965, 1966; Jaffe, 1969; Liener and Kakade, 1969; Liener, 1975). Trypsin inhibitors, which can inhibit the action of the enzyme trypsin, are believed to stimulate increased synthesis and flow of pancreatic digestive enzymes into the intestine (Stein, 1976). As a 13 result of increased protein synthesis, the pancreas may become enlarged, with a subsequent loss of essential amino acids (Liener, 1962). It has been demonstrated that approximately 40% of both the pancreatic hypertrophy and growth depression effects associated with raw bean consumption may be accounted for by trypsin inhibitor activity (Stein, 1976). Kakade and Evans (1965) reported that autoclaving navy beans at 1210 C for five minutes destroyed 80% of the trypsin inhibitor activity and the growth performance of rats improved markedly. Honavar et al. (1962) found that protein fractions of kidney beans which contained no antitryptic activity but which were high in hemagglutinating activity also inhibited the growth of rats when incorporated into basal diets containing 10% casein at levels as low as 0.5%. When the purified hemagglutinin was boiled for 30 minutes before being incorporated into the diet at 0.5%, no growth inhibition was observed. The findings of several workers including Kakade and Evans (1965,1966), Honavar et al. (1962), Iyer et al. (1980), and Thompson et al. (1983) indicate the importance of adequate heat treatment in order to eliminate the toxicity of trypsin inhibitors and hemagglutinins in P, vulgaris. However, there is some disagreement as to whether or not soaking, as well as heating, is required to completely inactivate these factors. Honavar et al. (1962) found that feeding weanling rats diets containing black or kidney beans which had been autoclaved caused the rats to grow very slowly or actually lose weight, whereas beans which had first been soaked and then autoclaved produced a growth rate comparable to that obtained when casein was fed. Although Kakade and Evans (1966) found that soaking navy beans 14 for 1 to 4 days decreased the trypsin inhibitory activity and especially the hemagglutinating activity, rats fed a diet of unsoaked, heat treated navy bean meal supplemented with methionine grew as well as rats fed a casein diet. More recently, Iyer et al. (1980) reported that soaking alone decreased the trypsin inhibitor activity of .3. vulgaris by only 15% or less, whereas soaking followed by convential cooking reduced the activity by approximately 90% of that present in the raw state. Thompson et al. (1983) found that presoaking red kidney beans followed by heating at 1000 C decreased hemagglutinin activity below detectable levels, as did pressure cooking without presoaking. Unlike the findings of Kakade and Evans (1966), these workers found that merely soaking the beans did not result in any loss of hemagglutinating activity. Thus, while heat treatment without prior soaking may not result in complete destruction of these antinutritional factors, it appears that any toxic effects are substantially reduced by heating alone. In general, the extent of the enhancement of nutritive value which results from heat treatment will depend on the temperature, length of heating, and moisture conditions (Liener, 1975). Phytic Acid In addition to the antinutritional factors already mentioned, legumes may contain relatively large amounts of phytic acid, up to 5% by weight (deBoland et al., 1975), which has the ability to bind nutritionally important mono- and divalent cations to form the complex phytate. Each phytic acid molecule contains 12 replaceable hydrogen atoms with which it may form insoluble salts with various elements. 15 The results of many studies conducted with several species indicate that the minerals present in phytate complexes become unavailable or only partially available (Sathe and Krishnamurthy, 1953; O'Dell and Savage, 1960; Roberts and Yudkin, 1960; O'Dell, 1969; O'Dell et al., 1972; Davies and Nightingale, 1975; Maga, 1982). The exact chemical structure of phytic acid, or mygfinositol hexaphosphoric acid [1,2,3,4,5,6-hexakis (dihydrogen phosphate)] has been debated for some time. The structure proposed by Anderson (1914) shown in Figure 1, suggests a symmetrical hexaorthophosphate 31P nuclear arrangement which has more recently been supported by magnetic resonance (Johnson and Tate, 1969). However, there is some evidence that phytic acid is present as the asymmetrical hydrated triphosphate structure proposed by Neuberg in 1908 (Oberleas, 1973). As such, Erdman (1979) hypothesized that both forms may exist, however it appears that the Anderson form predominates in plants. While several isomers of inositol hexaphosphate are known to exist and have been isolated from soils, only the HEB form has been isolated from plants (Cosgrove, 1966). Approximately 70% of the total phosphorus in dry beans is reported to be present as phytic acid or its calcium, magnesium, and potassium salts (Makower, 1969; Lolas and Markakis, 1977). Although several possible roles have been suggested for phytic acid, it is generally recognized that its primary function is to serve as a storage form of phosphorus to be used during germination (Oberleas, 1971; Lolas and Markakis, 1975; Erdman, 1979). It is thought that the formation of phytic acid prevents the accumulation of excessively high levels of inorganic phosphate (Erdman, 1979). The existence and 16 concentration of phytic acid or phytate vary substantially depending on which part of the plant is considered and its stage of maturity (Oberleas, 1973). 9’03“: Figure 1. Structure of phytic acid proposed by Anderson (1914). Using the increasing percentage of total solids as an indicator of increasing maturity, Makower (1969) measured changes in phytic acid in maturing pinto beans. During the early period of growth, characterized by enlarging cotyledons and pods, only a 14% change in total solids occurred, but phytic acid content increased from 0.13 to 0.77% of the dry bean weight. Most of the phytic acid was found to accumulate towards the end of maturation but before the rapid increase in the percent total solids. The increase in phytic acid phosphorus as a percentage of total phosphous was small after the midpoint in seed dry weight was obtained, indicating that little synthesis of 17 phytic acid occurred just prior to and during desiccation. Walker (1974) found that 90% of the total phytic acid in _P. vulgaris cotyledon was laid down between days 24 and 30 of the embryogeny process which normally requires 36 days. Rapid cotyledon enlargement was noted between days 12 and 30 and dehydration became apparent around day 32. Thus the findings of both workers indicate that most of the phytic acid accumulates towards the end of maturation, but before desiccation. In addition, Walker (1974) demonstrated that 90% of the phytic acid of P. vulgaris is lost within the first 10 days of germination. Although the precise location of phytate in legume seeds has not been determined, it is believed to be associated with protein (Oberleas, 1973). The phytate of most plant seeds is primarily contained in the bran and germ (Oberleas, 1973). When phytic acid is associated 'with the specific components as such, it may be preferentially extracted with them (Erdman, 1979). Ferrel et al. (1969) pointed out that in milling, phytate may become concentrated in high protein flours by the remilling of selected by-products. Wheat protein concentrate was found to contain approximately 20 times more phytic acid than wheat flour (Ranhotra, 1972). The phytic acid content of mature E. vulgaris has been measured by Lolas and Markakis (1975), Iyer et al. (1980), and Deshpande et al. (1982). For the fifty varieties of mature dry beans analyzed by Lolas and Markakis (1975), phytic acid content was found to range from 0.54 to 1.58%. Total phosphorus, which ranged from 0.259 to 0.556%, was found to correlate well (r=0.9847) with phytic acid content. Additionally it was found that over 99% of the total phytic acid 18 present in the beans analyzed existed in a water soluble form. The phytate phosphorus content of raw great northern, kidney, and pinto beans was reported to be 4.6, 5.8, and 5.5 mg/g, respectively, by Iyer et al. (1980). Based on the assumption that phosphous accounts for 28.2% of the weight of phytic acid, that represents an average of 1.8% phytic acid for the three bean types. Deshpande et al. (1982) measured the phytic acid content of 10 cultivars of dry beans for whole intact beans and dehulled and hull bean fractions to determine the effect of seed coat removal on phytic acid concentration. The phytic acid content of whole beans ranged from 1.16 to 2.93%, values almost twice that reported by Lolas and Markakis (1975). The authors attributed the discrepancies to differences in varieties of beans grown, climate, soil, location, irrigation conditions, and year. Except for pinto beans, dehulling increased the percentage of phytic acid by an average of 31% as compared to the whole beans. The researchers conjectured that phytic acid nay be characteristically present in the cotyledon fractions and also, since the seed coat contributes a relatively large portion of the whole seed weight, dehulling may result in an increase in the phytic acid concentration on a unit weight basis. It was suggested that the large increase in phytic acid coincident with hull removal might reflect an improvement in the extraction efficiency of this compound. Phytase Phytase enzymes which hydrolyze phytic acid to inositol and phosphoric acid have been identified in both plants and the intestinal tracts of several animal Species (Cosgrove, 1966; Johnson 19 and Tate, 1969; Bitar and Reinhold, 1972). While phytase is present in dry, dormant, mature seeds, the findings of several workers including Gibbons and Norris (1963), Makower (1969), and Walker (1974) indicate that it does not become active until germination. Lolas and Markakis (1977), Chang and Schwimmer (1977), and Gibbins and Norris (1963) isolated the phytase enzyme from P. vulgaris and demonstrated Optimal activity at pH 5.2 or 5.3. Whereas Lolas and Markakis (1977) reported 500 C to be the optimum temperature for phytase activity of navy beans, Chang and Schwimmer (1977) reported 600 C as optimal for California small white beans. In addition, Lolas and Markakis (1977) noted that almost complete inactivation of phytase occurred at BDC’C. Previously, optimal pH values of plant phytases have been reported to range from 4.5 to 5.5 with Optimal temperatures between 45 and 560 C (Sloane-Stanley, 1961). Phytase activity has been found to be inhibited by high substrate concentrations (Gibbins and Norris, 1963; Lolas and Markakis, 1977; Chang and Schwimmer, 1977) and by phytate- precipitants such as Cu +2, Zn +2, Fe+3, and Ca+2(Sloane-Stanley, 1961). Phytase activity has been demonstrated in the small intestine of the rat, chicken, calf, and man (Bitar and Reinhold, 1972), however little is known about the role of these intestinal phytases in phytate- phosphorus metabolism. Unlike trypsin inhibitors and hemagglutinins, phytates are relatively heat stable. Rackis (1974) reported that four hours of autoclaving at 115()C were necessary to destroy the majority of the phytic acid present in soy isolate. Such a long heat treatment would not be feasible since it would result in amino acid destruction as well. Using approximately the same temperature and time conditions, 20 Lease (1966) determined that only 20% of the phytate associated with sesame meal was destroyed. The findings of these workers support the conclusion reached by deBoland et al. (1975), that the thermal destruction rate of phytate appears to be product specific. Destruction of phytate has been reported for food systems in which phytase has been activated. Both rye flour and yeast are known to have relatively high phytase activities (Hoff-Jorgensen et al., 1946). During the fermentation of bread doughs containing rye flour and/or yeast, the phytase becomes activated, resulting in baked bread with a reduced phytate content (Reinhold, 1971). Metallic-Phytates Whether or not a specific mineral binds with phytic acid depends on pH as well as on the presence of secondary cations (Oberleas, 1973). Ferric and scandium phytates are least soluble in dilute acid and dissociate in concentrated acid or dilute alkali. Zinc and copper phytates are most stable between pH values of 4 and 7, and calcium, magnesium, and barium phytates form most readily under slightly alkaline conditions. Vohra et al. (1965) studied the titration curves of phytate as the free acid and as the sodium salt in the presence of various single polyvalent cations. These workers found that at pH 7.4, phytate formed complexes with metals in the following decreasing order: Cu+2 > Zn+2 > Co+2 > Mn+2 > Fe+3 > Ca+2. Zinc and copper formed the most stable metal-phytate complexes. When two or more cations are present simultaneously, they may act in unison to increase the quantity of metallic phytate precipitated 21 (Oberleas, 1973). Most notably this has been observed for zinc and calcium, and for copper and calcium. For example, doubling the molar concentration of calcium increased the percentage of calcium and zinc recovered in the phytate precipitate from 63 to 84% and from 67 to 97%, respectively. The ability of metallic phytates to depress absorption of the chelated minerals depends on many factors including the intestinal and food/meal phytase activities, previous food processing conditions (especially pH), digestibility of the food eaten, and the physiological status of the individual consuming the food (Erdman, 1979). The major adverse effect of phytic acid on mineral availability has been demonstrated in relation to zinc. The work of several researchers including O‘Dell and Savage (1960) and Likuski and Forbes (1965) has indicated that an inverse relationship exists between the level of phytic acid in the diet and zinc bioavailability. In addition, high levels of calcium associated with phytate have been shown to decrease zinc bioavailability (Oberleas et al., 1966). Davies and Nightingale (1975) found similar interactions betwen zinc and copper. Several reports which appeared in the literature during the 19305, 19405, and early 19505 indicated that cereals had anticalcifying and rachitogenic properties. Phytates were implicated as the causative agent based on findings of research conducted on dogs, however, in humans these results could not be substantiated (Maga, 1982). Oberleas (1973) noted that calcium absorption is influenced by vitamin D, lipids, and other dietary factors in addition 22 to dietary phytate. Therefore foods having a calciumzinorganic phosphate ratio between 1:1 and 2:1 and that contain adequate amounts of calcium, phosphate, and vitamin D, in all liklihood will not be rachitogenic even if calcium is bound by phytate (Oberleas, 1973). Roberts and Yudkin (1960) cited dietary phytate as a possible cause of magnesium deficiency and later it was shown by Likuski and Forbes (1965) that calcium in conjuction with phytic acid may also depress magnesium absorption. In addition, phytate phosphorus is generally considered to be unavailable for monogastric animals including man (Oberleas, 1973). Next to zinc, iron is the nutritionally important mineral most frequently associated with phytate binding. Although iron availability in relation to phytic acid has been studied by several researchers, the data are not in agreement as to whether phytate inhibits iron absorption or has no effect. Nutritional Significance of Iron-Phytate Iron Absorption, Transfer and Storage The basis for phytic acid inhibiting iron absorption in humans relates to the insolubility of iron phytate at the exceptionally low pH provided by the gastric juice of the stomach. It is thought that since iron phytate is not solubilized by the gastric juice, it does not ionize, and therefore cannot be absorbed. As such, the effect of phytic acid on mineral availability, if any, is as an intraluminal factor which inhibits solubilization of iron before the ingested food leaves the stomach (Prasad, 1978). Normally, the iron from foods and iron salts is solubilized and 23 ionized by the gastric juice and passes from the stomach to the duodenum where the majority of iron absorption occurs (Underwood, 1977; Narasinga Rao, 1981). In the duodenum, the pH increases from approximately 1.5 to 7.0 as a result of duodenal secretions, causing the precipitation of ferric (Fe+3 ) ions, but maintaining the solubility of the ferrous (Fe+2) form. Thus it is generally accepted that ferrous iron is better absorbed than ferric forms (Narasinga Rao, 1981). The amount of iron which is transferred from the gut lumen to the mucosa depends on the number of receptors on the brush border; the number increasing during iron deficiency. While the inorganic iron supplied by food and/or inorganic iron salts (e.g. iron supplements) must be taken up by the brush border receptors, heme iron (ingested as such or made available by the removal of nonviable red blood cells) enters the mucosal cells directly without first having to be released from the bound form. Once in the mucosal cell, xanthine oxidase liberates iron from the heme complexes (Prasad, 1978; Narasinga Rao, 1981). From the mucosal cell, iron is transferred to plasma transerrin which carries ferric iron to the bone marrow for hemoglobin (Hb) synthesis, or to reticuloendothelial cells for storage as the two nonheme compounds ferritin and hemosiderin (Underwood, 1977; Beutler, 1980; Narasinga Rao, 1981). Once in the bone marrow, ferric iron is reduced to the ferrous form and detached from transferrin, facilitating its transfer to protoporphyrin such that it becomes stabilized in Hb, a complex of globin and four ferroprotoporphyrin moities, where it can reversibly bind to oxygen, permitting Hb to 24 serve as an oxygen carrier (Underwood, 1977; Prasad, 1978; Beutler, 1980). A small amount of iron is also incorporated in various enzymes as iron-porphyrin complexes, iron-flavoproteins, or it may serve as a cofactor. The total amount of circulating Hb, approximately 800 to 9009 in an average man, is synthesized and catabolized every 120 days. The breakdown of red blood cells releases about 20 mg of endogenous iron each day, which is reutilized for H6 synthesis (Prasad, 1978). Over 90% of body iron is conserved and reutilized (Beutler, 1980). Ferritin, the water soluble storage form of iron, constitutes a slowly exchangeable pool of iron which can be mobilized by exchanging with the carriers. As the level of iron to be stored increases, more iron is stored as the insoluble hemosiderin. Both storage forms of iron serve as reserves to protect against sudden losses due to unanticipated blood loss (Prasad, 1978; Tyler, 1979; Narasinga Rao, 1981). Other Factors Influencing Iron Absorption Since the body has only a limited capability to excrete iron, homeostasis of this nutrient is maintained primarily by regulation of the amount absorbed. Healthy individuals are estimated to absorb 5 to 10% of dietary iron whereas for iron-deficient individuals it is believed that this amount ranges from 10 to 20%. Iron deficiency nay result in the develOpment of hypochromic, microcytic anemia accompanied by a normoblastic, hyperplastic bone marrow that contains little or no hemosiderin. The existence of iron deficiency without anemia has also been confirmed by several investigators and is believed to be two to three times more prevalent than true iron 25 deficiency anemia (Prasad, 1978). Evidence exists which suggests that some of the symptoms of anemia may actually be due to decreased activity of intracellular enzymes, and not to low levels of Hb, as previously thought (Tyler,1979). In addition to phosphates and phytic acid, several factors are known to influence iron absorption. The presence of other foods and food components in the diet, particularly meat and fish, but also tea, dietary fiber, fat, and ascorbic acid have been reported to influence iron absorption (Young and Janghorbani, 1981). Layrisse et al. (1969) investigated the availability of iron from various food sources with 5Fe or 59Fe in both iron adequate and deficient human subjects. These workers found absorption to range from 2% for lettuce to 20% for veal. Relatively low mean absorption values of 1.7 to 7.9% were reported for wheat, corn, black beans, lettuce, and spinach. Martinez-Torres and Layrisse (1970) observed that iron absorption in human subjects from either corn or black beans administered as a test meal was enhanced when these foods were mixed with veal, fish, or a mixture of amino acids similar to that present in fish. Based on the percentage transferrin saturation values, iron absorption in 18 healthy adults from black beans alone was 1.3%, but this amount increased to 3.1% when the beans were administered with amino acids or fish. 0f the amino acids fed, only cysteine was found to enhance absorption. Although it is a common notion that iron from animal foods is better utilized than that from plant sources, the findings of Fritz et al. (1970) could not confirm this. These workers conducted repletion tests with both weanling rats and chicks to compare the availability of iron from animal sources including blood meal, egg yolk, and fish 26 protein concentrate with plant sources including biscuits, corn meal, corn germ, cereal, and various flours. Perhaps the specific selection of animal and plant foods used in the investigation was not representative of all animal and plant foods which are consumed. Amine and Hegsted (1971), in working with iron deficient rats, found that as the level of salt/mineral mix in the diet was increased, iron absorption decreased. Iron retention, as measured by the difference in total body count two hours and nine days after ingestion of 59Fe, was greatest in rats fed diets containing no salt/mineral mix. Retention was significantly lower from the diet supplying 2% salt/mineral mix than from the diet containing no salt/mineral mix. Similarly, iron retention was significantly higher from the diet containing 2% salt mix than the one containing 4% salt mix. When the carbohydrate source of the diets was changed, iron retention was significantly lower from diets containing glucose than from those containing 60% sucrose, but significantly higher from glucose than from diets containing 60% starch. In terms of the carbohydrate source, maximum retention was observed with diets containing 60% lactose or 20% lactose and 40% starch. Reducing agents such as cysteine, fructose, and glutathione are thought to enhance iron absorption by reducing Fe +3 to Fe +2 , thus preventing precipitation at the neutral or alkaline pH of the duodenum (Narasinga Rao, 1981). The absorption of inorganic iron is much more sensitive to changes in the intestinal environment than is the absorption of heme iron. Unlike nonheme forms of iron, heme iron absorption is not increased by ascorbic acid or decreased by phytates (Underwood, 1977; Beutler, 1980). 27 Feeding Studies Relating Iron Absorption to Phytic Acid Intake Sathe and Krishnamurthy (1953) placed anemic young rats (Hb = 7-8 g/IOOmL) into three groups of five rats each and fed them semi- purified diets containing rice which received no polishing, slight polishing, or much polishing, corresponding to high phytate, moderate phytate, and low phytate diets. Iron absorption as measured by hemoglobin gain was determined after one, two, three, and four weeks. These workers attributed the differences in hemoglobin levels obtained for the three groups to differences in the phytic acid content of the diets, such that hemoglobin levels were higher for rats fed diets containing less phytic acid. Using healthy adolescent boys as their subjects, Sharpe et al. (1950) measured iron absorption as retention of 55Fe or 59Fe from seven different breakfasts chosen to contain various amounts of phytic acid. The iron content of each breakfast was standardized using ferric chloride. One breakfast consisted solely of water and ferric chloride, another of milk plus added ferric chloride and one of milk, sodium phytate, and ferric chloride. The other four breakfasts contained one or more of the following in varying amounts: cooked rolled oats, white bread, egg omelet, and tomato juice. The authors found iron absorption to be greatest from the distilled water, whereas milk alone, which contained no phytate, appeared to decrease iron absorption appreciably. It was expected that the rolled oats would have a high phytate content and therefore would depress iron availability. However, the rolled oats and milk together reduced absorption by only twice as much as milk alone, and no correlation was 28 found between the phytate content of the oats and the reduction of iron absorption. Therefore these workers, unlike Sathe and Krishnamurthy (1953), concluded that endogenous phytate was not an important factor in decreasing iron absorption. Sharpe et al. (1950) hypothesized that the calcium in milk may have preferentially combined with phytate, thus iron availability was not affected to a greater extent. Added sodium phytate, however, was found to decrease the absorption of iron by 15 fold, indicating that added soluble phytates could interfere with iron absorption. Similarly, studies conducted by Hussain and Patwardhan (1959) with four healthy male subjects revealed that sodium phytate added to otherwise adequate diets inhibited iron absorption. Diets were prepared to contain 8% and 40% of the total phosphorus from phytate, and equivalent amounts of endogenous iron. Little differences in individual and average iron intakes were noted on both diets, however iron retention of subjects on the diet containing 40% phytate phosphorus was markedly reduced. 0n the 8% phytate ph05phorus diet, more than 10% of the dietary iron was absorbed. At the 40% level, absorption was less than 3%. Unlike the findings with human subjects which demonstrated inhibition of iron absorption from added sodium phytate, Cowan et al. (1966) found that varying levels of added sodium phytate had no effect on iron absorption by anemic rats. Anemic Sprague-Dawley rats (Hb < 7 g/IOO mL) were distributed into groups of eight and for four weeks were fed purified diets containing 10 or 20 ppm iron as ferous sulfate with either 0, 45, or 75% of the phosphorus derived from sodium phytate. Expressed in terms of milligrams of iron consumed, there 29 were no significant differences at either the 10 or 20 ppm level of iron between control values and those of the diets containing sodium phytate. The authors concluded that high levels of phytate have no effect on iron absorption in the rat. Hunter (1981) confirmed the findings of Cowan et al. (1966) in a similar experiment. Hunter (1981) fed iron deficient rats one of 16 diets containing either 0, 4, 8, or 12 ppm iron as ferrous sulfate and O, 0.25, 1.0, or 4.0% sodium phytate. As measured by Hb regeneration after two weeks, iron absorption from any of the variable phytate diets was not significantly different from the iron absorption by rats fed the phytate free diet. Hunter (1981) also force fed both iron deficient and iron adequate rats slurries containing an iron deficient nge, and sodium phytate to compare the amount of iron absorbed diet, by the iron adequate and iron depleted rats. Iron absorption, measured as the percentage of the 59Fe dose retained after nine days, was greater for the iron deficient rats. The addition of sodium phytate depressed iron absorption by about 30% in the iron adequate rats but by only 18% in the iron deficient rats. This finding is consistent with the basic principle of maintaining iron homeostasis by regulating iron absorption. Instead of studying the effects of added phytate, Welch and VanCampen (1975) were interested in determining the effects of endogenous phytic acid in soybean seeds on the bioavailability of 59Fe to iron-depleted rats. Both immature and mature soybean seeds containing Fe were fed in a single dose to young male rats (Hb 59 averaging 8.3 g/100 mL). As measured by whole-body counting, Fe 5 from the mature soybean seeds was more available than the Te present 30 in the immature seeds even though the phytic acid content of the mature seeds was almost three times that of the immature seeds. The authors concluded that the availability of 59Fe from soybean seeds was not directly correlated to their phytic acid content. In addition they hypothesized that the immature seeds might contain another factor which could account for the reduced iron availability. Also studying the effect of endogenous phytate on iron absorption, Ifon (1981) fed young iron depleted rats (Hb averaging 9 g/100 mL) semipurified diets containing 20 ppm iron from millet, guinea corn, maize, soybeans or bambara nuts. No correlation was found between the level of ingested phytate and the proportion of iron utilized for Hb synthesis. Maize and bambara nuts, both of which had high phytic acid contents, showed high levels of iron utilization. Thus it was concluded that the effect of phytic acid on iron absorption was minor or nonexistent. Morris and Ellis (1976) isolated monoferric phytate from wheat bran and found it to be a water soluble complex of iron which had no negative effect on iron absorption and was actually of high biological availability. Iron availability as measured in a hemoglobin repletion test with iron depleted rats revealed that the monoferric phytate afforded the same biological availability as ferrous ammonium sulfate, which was the reference compound used. In contrast, ferric phytate, which contains three to four moles of iron per mole of phytate was found to be insoluble and of low bioavailability. These researchers considered monoferric phytate to be the predominant form of iron in wheat, being bound to cationic sites of protein or other cellular constituents of the wheat bran. The authors conjectured that seemingly 31 conflicting reports in the literature on the effect of phytate on iron absorption might be due to the formation or use of ferric phytates of differing degrees of saturation. Other recent research indicates that decreased iron availability once attributed to phytate may be due to binding with fiber or other factors. Reinhold et al. (1975) dephytinized wholemeal breads and separated zinc, calcium, and iron and found that the binding of these minerals increased after phytate removal due to increased fiber concentration. These workers concluded that fiber and not phytate was the primary determinant of the availability of the divalent minerals in wholemeal bread. Morris et al. (1980) prepared muffins with dephytinized wheat bran (DWB), whole wheat bran (WWB), and without bran (NBM) and fed them with a standard meal (STD) of cooked beef and a milk shake to healthy human subjects. WWB decreased nonheme iron absorption by 2.4 fold when the STD included ascorbic acid and four fold when meat or ascorbic acid were omitted. The absorption ratio of STD and NBM to STD and WWB or DWB was 2.4 and 1.8, respectively, indicating that the inhibitory effect of bran was not solely due to the phytate content. Insoluble, high fiber fractions of DWB were also found to inhibit absorption of inorganic iron. Thus, it can readily be seen that the complex interaction of many factors makes the prediction of iron bioavailability difficult. As such, the possible significance of only phytic acid in relation to iron nutrition in humans is difficult to determine. When reviewing the findings of research or planning investigations relating phytic acid to iron availability, it is important to consider the composition 32 of the total diet and to remember that animal models, especially in iron deficient states, may not necessarily provide precise estimates of the true availability of minerals from actual diets consumed by healthy human beings under normal living conditions. MATERIALS AND METHODS Raw Material Procurement and Processing Source Prime, handpicked navy, pinto, and black beans (Phaseolus Vulgaris L.) from the 1981 Michigan crop were used in the production of dry roasted fractionated bean flours. Ninety-one kilograms of each type of bean were shipped to the Food Protein Research and Development center at Texas A&M University for processing. Roasting and Dehulling Forty-five kilograms of each type of bean were roasted in a particle-to-particle heat exchanger (custom built by Food Processes Inc., Saginaw, MI). The heat transfer medium of the exchanger consisted of 1.6 mm (1/16") diameter,type A, 90% aluminum oxide ceramic beads (Coors Ceramic Co., Golden City, CO) with a specific gravity of 3.6 g/cm3 . The beads were heated to 2400 C and were maintained in the chamber with the raw beans for 100 seconds in a 1:5 ratio of beans to beads. These processing conditions resulted in an exit temperature of the beans of 113C’C. Roasted beans were cracked through a corrugated roller mill (Ferrell Ross; Oklahoma City, OK) into 6 to 8 pieces. The hulls, or seed coats, were removed using a zig-zag aspirator (Kice Metal Products; Wichita, KS). A hull flour fraction was produced by grinding the hull pieces through a swinging 33 34 blade Model 06 Fitzmill (W.J. Fitzpatrick Co., Chicago, IL) using the impact surfaces for pulverization through a 0.69 mm (1/37 inch) round hole screen. Grinding and Air Classification After hull removal was completed at the Food Protein Research and Development Center, the cracked cotyledons were sent to the Alpine American Corporation; Natick, MA for milling and air classification. The cracked cotyledons were finely ground in a Model 250 CW Stud Impact Mill at a speed of 11,789 rpm and a door speed of 5,647 rpm. The resulting flours were air classified in a Model 410 MPVI Air Classifier at a rotor speed of 2,200 rpm and a brake ring setting of 3, using a 7.62 cm (3 inch) screw feeder operating at 25 rpm. At this point, two flour fractions were obtained: an intermediate starch fraction (coarse I) and an intermediate protein fraction (fines I). The intermediate protein fraction was reclassified under the following conditions: rotor speed of 2,200 rpm; brake ring setting of O; 7.62 (3 inch) screw feeder operating at 25 rpm. As a result of this second air classification step, a high starch fraction (coarse II) and a high protein fraction (fines II) were obtained. Sample Materials The final materials produced as a result of the processes described included the hull flour fraction and three air classified fractions: intermediate starch (starch I), high starch (starch II), and high protein (protein 11). A flow diagram showing the flour processing scheme and the fractions produced appears in Figure 2. The percentage yield of each fraction of each bean type is shown 35 52“. 522a :9: 52”. 655 :9: = 2.505 __ +6me _ Z_m._.Omn_ _ ION—$5.. 02.1.1...)— Z_n_ wZODMA>POO .2553 9.3383 .52.. .8 5852.. so: .m 8.sz £304... 1.151 - ozaon - min: zo_+> 36 diagramatically in Figure 3. In addition to these four flour fractions, a small amount of dehulled, ground cotyledon material, referred to as the whole-dehulled fraction, was reserved and later analyzed. All flour fractions were packaged in polyethylene bags and shipped to Michigan State University in fiber drums at ambient temperature. Upon arrival at Michigan State, within two weeks after shipping, the flour fractions were maintained in the shipping containers at room temperature. The five flour fractions of each of the three bean types were employed in all subsequent analyses. Proximate analyses were begun immediately upon arrival. Mineral and phytic acid analyses were conducted within the following four months. Proximate Analyses Moisture AACC Method 44-32 (1962) was modified slightly for determining moisture content in duplicate. Five 9 flour samples were weighed into previously dried, cooled, and weighed 50 ml porcelain crucibles. Samples were dried under a partial vacuum (ca. 25 mm Hg) between 95 and 1000 C for 6 hours. After cooling in a desiccator, samples were reweighed and percentage moisture was calculated as follows: Wet sample wt. - Dry sample wt. % Moisture = X 100 Wet sample wt. Ash Ash content was determined for each flour fraction in triplicate by AACC Method 08-01(1962). The dried flour samples obtained from the moisture determinations were incinerated at 5250 C for 24 hours in a .mcoma xum~n can .ou:_a .>>m: scum umcwmuno mcowuomcm czopm mo ope?» mmaucmucoa .m «camp; Z_m._.Omn_ I n. = ION—$5.. I Nlm . Iom>mc msovcm> mo cowpwmoaeoo mumsrxoca .v mcamwd .amaaaxun. 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N.N2m:.s Hcmwm mo ucmpcoo me2m>< .m mpnmh 59 This finding was consistent with the generally higher ash values reported for the protein fractions, and consistently low ash values reported for the starch II fractions. Calcium The average calcium content of the pinto flour fractions was consistently lower than for the corresponding navy and black flour fractions. Values ranged from 216 to 2433 ppm for pinto flours, from 273 to 4173 ppm for navy flours, and from 696 to 4270 ppm for black flours. For all bean types, the starch II fraction contained the least calcium. For both navy and black bean flours, the hull fraction contained the most calcium, whereas the starch I fraction of the pinto flour had the highest calcium content. Copper Average copper values ranged from 9.6 to 52.6 ppm for navy flours, from 8.5 to 67.2 ppm for pinto flours, and from 4.9 to 16.5 ppm for black flours. The lowest values were found for the starch II fraction of each bean type. The starch I fraction of navy and pinto flours contained the most c0pper. For the black flours, the hull fraction had the highest copper value, however, this amount was not significantly different from the copper content of the protein fraction, which differed from the hull fraction by less than one ppm. Each fraction of black flour contained less capper than the corresponding navy and pinto flours. 60 .mco_pum22 230.2 :mwn xumpn new oucwa .x>mc mzow2m> $0 “cupcou anopmu mmm2m>< .N m2smwu ¥O>mc mzow2m> 2o ucwucou 2manou mmm2w>< ¥0<4m 05.2: 2.3.0.... D .. 3052.6 % . 20.2.5 I .15: g Guadazmo INJOIB a l. I! W lilli l Hill!!! I IN! iiiliiiilllllllliiéliél I >>>0: 000w2m> 20 0:00:00 :02. mmm2m>< .0 0200.0 um. 00. 00« Au 000 d m 2.30.... D 00.. : :05; m . 30530 I .30: g 000 000 Omddazma -30.; EB 64 .m:0..002. 200.. :00: 200.: 0:0 00:.0 .x>0: m:0.20> .0 0:00:00 20.00:mms 0mm20>< .0. 0200.: 20(40 05.2.1 >>>0: 000.200 .0 0:00:00 0020000000 00020>< ¥0<4m _ 05.2...— >>>0: m00.20> .0 0:00:00 0:.N 0m020>< .N. 0200.. ¥O<4m 0.5,...— >>0: 000.20> .0 0:00:00 50.000 00020>< 2.000.... n”— : :00<..0 m . 2052.0 m .30: g amaaazmn -Sozsg .m. 0200.. 00 00 00. d w 00. .0nuu 69 .0:0.0002. 200.. :000 000.0 0:0 00:00 .x>0: 000.20> .0 0:00:00 50.000000 0m020>< .0. 0200.0 204...; 0.5.... >>0: 000.20> .0 0:00:00 0.00 0.0000 00020>< .0. 0200.0 200..—m, 00.2.0. >>0= 0000000000 00000 0000 000 00—0 000— :0. .0000 000 0000 0.5000 00 0000020000 000000000 .00 0—000 87 Table 17. Iron content of test and standard diets determined in repeated analyses. parts per million (ppm) Diet Analysis 11 Analysis 22 Analysis 33 6.3 1.80 9.23 Basal low iron 7.8 1.90 9.92 Ferrous sulfate-6 ppm 5.7 3.40 14.40 6.4 3.20 14.89 9.2 - 13.09 Ferrous sulfate-12 ppm 14.9 4.40 26.86 7.3 4.40 19.31 4.4 - 15.85 Ferrous sulfate-24 ppm 18.2 6.70 57.83 16.0 12.20 49.27 11.3 - 29.19 Navy hull flour-6 ppm 5.5 7.70 19.75 3.2 6.60 21.03 5.7 - 18.29 Navy hull flour-12 ppm 9.3 11.50 28.42 9.2 11.41 34.68 7.3 - ‘26.24 Navy hull flour-24 PPm 10.5 20.79 52.58 12.1 21.90 71.55 7.9 - 49.62 Navy starch II flour 10.3 5.60 14.30 6 ppm 5.8 5.20 15.84 7.6 - 16.75 Navy starch II flour 11.0 8.70 24.32 12 ppm 11.4 6.50 26.80 12.8 - 23.06 Navy starch 11 flour 25.0 17.70 29.68 24 ppm 22.4 15.11 34.54 21.6 - 35.01 Navy protein flour-6 ppm 15.0 7.20 22.42 10.4 7.30 24.03 12.3 - 22.77 Navy protein flour-12 ppm 21.1 20.10 42.70 25.2 21.29 46.69 23.4 - 44.02 Navy protein flour—24 ppm 35.6 23.89 57.66 35.4 21.60 58.53 41.2 - 63.07 1Values were determined for dry ashed samples by plasma emission. 2Values were determined for dry ashed samples by atomic absorption. 3Values were determined for wet ashed samples by atomic absorption. 88 groups consuming diets containing different flours, even at the same iron level. Per milligram of iron consumed, groups fed protein flour diets consumed more phytic acid than groups fed hull flour diets. Groups fed hull flour diets consumed more phytic acid than groups fed starch II flour diets per milligram of iron consumed. The weight gain data reveals that groups fed diets containing higher levels of iron gained more weight, however the total food intake values of these groups of animals were not substantially different from intake values of groups fed the basal diet or diets containing the lowest level of added iron. Presumably the reason for this relates to the accuracy of recording food consumption and not the actual amount of food eaten. The difficulty in recovering spilled feed could account for the discrepancy. The final Hb concentrations of the 13 groups indicate that true differences -did exist both between groups fed different amounts of iron and those fed iron from different sources. The group fed the basal, low iron diet showed the lowest final Hb value (3.9 g/100 ml). Comparison of groups fed ferrous sulfate with groups fed navy hull flour shows similar responses in terms of final Hb concentration at all levels. As such the effect of phytic acid on iron absorption from navy hull flour does not appear to be major. The final hemoglobin concentrations were markedly lower for the groups fed navy starch II than for the groups fed ferrous sulfate. Since the estimated iron intake of the group fed navy starch II at a level of 24 ppm was lower than the corresponding group consuming ferrous sulfate, it is difficult to establish to what extent, if any, phytic acid influenced iron absorption. A statistical procedure such 89 as a slope ratio or parallel lines assay should be conducted in order to ascertain this. Groups fed protein flour diets showed greater final Hb concentrations than groups fed ferrous sulfate diets. One possible explanation for this finding is that the protein flour diets contained more iron than was estimated. If that assumption is correct, unless the actual amount of iron present was much greater than the estimated amount, a reduction in iron absorption might still be expected owing to the relatively high phytic acid content of this flour fraction. Taking this into consideration, it appears likely that the phytic acid present did not inhibit iron absorption from navy protein flour. If the actual iron contents of these protein flour diets were very similar to the estimated values, the possibility of there being some factor present which could have enhanced iron absorptin might deserve investigation. Without having conducted the statistical analyses appropriate for this type of study, it is not correct to conclusively state that phytic acid did or did not influence iron absorption from the various bean flour fractions. However, it is within reason to state that a preliminary examination of the data suggests that the absorption of iron from navy bean flours by anemic rats does not appear to be adversely affected by the presence of endogenous phytic acid. SUMMARY AND CONCLUSIONS A major objective of this study was to demonstrate the partitioning of several nutritionally important minerals and phytic acid among hull, intermediate starch, high starch, high protein, and whole-dehulled flour fractions derived from navy, pinto, and black beans. To determine if endogenous phytic acid influenced the availability of iron from any of the flours, hemoglobin regeneration in anemic rats fed diets containing various bean flour fractions as the iron source was compared to the hemoglobin regeneration in rats fed standard diets containing ferrous sulfate. Results of the mineral analysis indicated that phosphorus, zinc, iron, potassium, and magnesium shifted into the protein flour fractions of all three bean types. For both navy and black bean flours, calcium became concentrated in the hull flour, presumably in association with the fiber component of this fraction. Copper and sodium showed no partitioning trends across bean types. Except copper and sodium, all minerals were present in the smallest amounts in the high starch flours. Small quantities of all minerals in the high starch fractions of all bean types indicated that these flours could not be considered good dietary sources of the minerals studied. Phytic acid content of the three bean types ranged from low values of 4.29 - 8.72 mg/g for the high starch flours to high values of 23.74 - 30.22 mg/g for the protein flour fractions. Thus, partitioning of phytic acid with the protein flour fraction was noted. 90 91 Correlation coefficients generated between phytic acid content and protein content, between phytic acid content and phosphorus, iron, zinc, magnesium, and potassium content, and between the content of these minerals and protein content were very strong and highly significant (p.g .01) for all three bean types. Based on these findings it was concluded that the phytic acid present in dry beans is associated with protein. As such, any processing techniques implemented to isolate protein will also serve to increase the concentration of phytic acid. The high degree of correlation achieved between protein content and the content of zinc, iron, potassium, and magnesium, and between phytic acid content and these minerals suggests that these elements were present as metallic phytates. Tbtal phosphorus content, of which phytate phosphorus is a major component, appeared to be a reliable indicator of the phytic acid content of various bean flour fractions. The data obtained from the feeding study indicates that rats fed navy hull, high starch, and protein flours showed changes in hemoglobin concentration comparable to those of groups fed the standard ferrous sulfate diets. Overall, without having conducted the appropriate statistical analyses, it appears that although iron may have been bound to phytic acid, its absorption by anemic rats was not hindered by the presence of endogenous phytic acid. In future investigations, the bioavailability of other minerals, especially zinc, from various bean flour fractions should be considered. Other nutritional aspects of air classified bean flours which warrant further study include the partitioning of water-soluble vitamins and amino acids among the various flour fractions. LIST OF REFERENCES LIST OF REFERENCES Aguilera, J.M., Lusas, E.W., Uebersax, M.A., and Zabik, M.E. 1982. Development of food ingredients from navy beans (Phaseolus vul aris by roasting, pin milling, and air classification. 3. F003 Sci. 47:1151. American Association of Cereal Chemists. 1962. AACC approved methods, 7th ed. The Association. St. Paul, Minnesota. Amine, E.K. and Hegsted, 0.M. 1971. Effect of diet on iron absorption in iron-deficient rats. J. Nutr. 101:927. Anderson, R.J. 1914. A contribution to the chemistry of phytin. J. Biol. Chem. 17:171. AOAC. 1980. “Official Methods Of Analysis." Section 43. 13th ed. Assoc. Offic. Agric. Chemists, Washington, D.C. Augustin, J., Beck, C.B., Kalbfleish, G., Kagel, L.C., and Matthews, R.H. 1981. Variation in the vitamin content of raw and cooked commercial Phaseolus vulgaris classes. J. Food Sci. 40:552. Baloorforooshan, M. and Markakis, P. 1979. Protein supplementation of navy beans with sesame. J. Food Sci. 44:390. Beutler, E. 1980. Iron. In "Modern Nutrition In Health and Disease." R.S. Goodhart and M.E. Shils, eds. Lea and Febiger, Philadelphia, Pennsylvania. Bitar, K. and Reinhold, J.G. 1972. Phytase and alkaline phosphatase activities in the intestinal mucosae of rat, chicken, calf, and man. Biochem. Bi0phys. Acta. 268:442. Bressani, R. 1975. Legumes in human diets and how they might be improved. In "Nutritional Improvement of Food Legumes by Breeding." M. Milner, ed. John Wiley and Sons, New York, New York. Cartland, G.F. and Koch, F.C. 1928. Alnicro-modification of the Keith-Rowntree plasma dye method for the estimation of blood volume in the rat. Am. J. Physiol. 85:540. Chang, R. and Schwimmer, S. 1977. Characterization of phytase of beans (Phaseolus vulgaris). J. Food Biochem. 1:45. 92 93 Charley, H. 1970. Legumes. In “Food Science." H. Charley, ed. John Wiley and Sons, New York, New York. Cosgrove, D.J. 1966. The chemistry and biochemistry of inositol polyphosphates. Rev. Pure Appl. Chem. 16:209. Cowan, J.W., Esfahani, M., Salji, J.P., and Azzam, S.A. 1966. Effect of phytate on iron absorption in the rat. J. Nutr. 111:841. Davies, N.T. and Nightingale, R. 1975. The effects of phytate on intestinal absorption and secretion of zinc, and whole-body retention of Zn, copper, iron and manganese in rats. Br. J. Nutr. 34:243. deBoland, A.R., Garner, 6.8., and O'Dell, B.L. 1975. Identification and properties of ”phytate“ in cereal grains and oilseed products. J. Agric. Food Chem. 23:1186. Deschamps, I. 1958. Peas and beans. In “Processed Plant Protein Foodstuffs." A.M. Altschul, ed. Academic Press, New York, New York. Deshpande, S.S., Sathe, S.K., Salunkhe, D.K., and Cornforth, D.P. 1982. Effects of dehulling on phytic acid, polyphenols, and enzyme inhibitors of dry beans (Phaseolus vulgaris L.). J. Food Sci. 47:1846. Erdman, J.W. 1979. Oilseed phytates: Nutritional implications. J. Am. Oil Chem. Soc. 56:736. Ferrel, R.E., Wheeler, E.L., and Pence, J.W. 1969. Phytic acid in millfeed by-products. Cereal Foods World. 14:110. Fordham, J.R., Wells, C.E., and Chen, L.H. 1975. Sprouting of seeds and nutrient composition of seeds and sprouts. J. Food Sci. 40:552. Fritz, J.C., Pla, G.W., Harrison, B.N., Clark, G.A., and Smith, E.A. 1978. Measurement of the bioavailability of iron, using the rat hemoglobin repletion test. J. Assoc. Off. Anal. Chem. 61:709. Fritz, J.C., Pla, G.W., Roberts, T., Boehne, J.W., and Hove, E.L. 1970. Biological availability in animals of iron from common dietary sources. J. Agr. Food Chem. 18:647. Gibbins, L.N. and Norris, F.W. 1963. Phytase and acid phosphatase in the dwarf bean, Phaseolus vulgaris. Biochem. J. 86:67. Hoff-Jorgensen, E., Anderson, 0., and Nielson, G. 1946. The effect of phytic acid on the absorption of calcium and phosphorus. Biochem. J. 40:555. 94 Honavar, P.M., Shih, C., and Liener, I.E. 1962. Inhibition of the growth of rats by purified hemagglutinin fractions isolated from Phaseolus vulgaris. J. Nutr. 77:109. Hunter, J.E. 1981. Iron availability and absorption in rats fed sodium phytate. J. Nutr. 111:841. Hussain, R. and Patwardhan, V.N. 1959. The influence of phytate on the absorption of iron. Ind. J. Med. Res. 47:676. Ifon, E.T. 1981. Bioavailability to rats of the iron contents in selected cereals and pulses. Nutr. Rep. Int. 24:25. Iyer, V., Salunkhe, D.K., Sathe, S.K., and Rockland, L.B. 1980. Quick-cooking beans (Phaseolus vulgaris L.): II. Phytates, oligosaccharides, and antienzymes. Qual. Plant Pl. Foods Hum. Nutr. 30:45. Jaffe, W.G. 1969. Hemagglutinins. In "Toxic Constituents of Plant Foodstuffs." I.E. Liener, ed. Academic Press, New York, New York. Johnson, L.F. and Tate, M.E. 1969. The structure of myg¢inositol pentaphosphates. Ann. N.Y. Acad. Sci. 165:526. Kakade, M.L. and Evans, R.J. 1965. Nutritive value of navy beans (Phaseolus vulgaris). Brit J. Nutr. 19:269. Kakade, M.L. and Evans, R.J. 1966. Effect of soaking and germinating on the nutritive value of navy beans. J. Food Sci. 31:781. Kay, D.E. 1979. Haricot bean. In "Food Legumes." D.E. Kay, ed. Tropical Products Institute, London, England. Koehler, H.H. and Burke, 0.W. 1981. Nutrient composition, sensory characteristics, and texture measurements of seven cultivars of dry beans. J. Amer. Soc. Hort. Sci. 106(3):313. Layrisse, M., Cook, J.D., Martinez, C., Roche, M., Kuhn, I.N., Walker, R.B., and Finch, C.A. 1969. Food iron absorption: A comparison of vegetable and animal foods. Blood. 33:430. Lease, J.G. 1966. The effect of autoclaving sesame meal on its phytic acid content and on the availability of its zinc to the chick. Poult. Sci. 45:237. Liener, I. 1975. Antitryptic and other antinutritional factors in legumes. In "Nutritional Improvement of Food Legumes by Breeding.“ M. Milner, ed. John Wiley and Sons, New York, New York. Liener, I.E. 1962. TOxic factors in edible legumes and their elimination. Am. J. Clin. Nutr. 11:281. 95 Liener, I.E. and Kakade, M.L. 1969. Protease inhibitors. In "Toxic Constituents of Plant Foodstuffs." I.E. Liener, ed. Academic Press, New York, New York. Likuski, H.J.A. and Forbes, R.H. 1965. Mineral utilization in the rat. IV. Effects of calcium and phytic acid on the utilization of dietary zinc. J. Nutr. 85:230. Lolas, G.M. and Markakis, P. 1975. Phytic acid and other phosphorus compounds of beans (Phaseolus vulgaris L.). J. Agric. Food Chem. 23:13. Lolas, G.M. and Markakis, P. 1977. The phytase of navy beans (Phaseolus vulgaris). J. Food Sci. 42:1094. Maga, J.A. 1982. Phytate: Its chemistry, occurence, food interactions, nutritional significance, and methods of analysis. J. Agric. Food Chem. 30:1. Makower, R.U. 1969. Changes in phytic acid and acid-soluble phosphorus in maturing pinto beans. J. Sci. Food Agric. 20:82. Makower, R.U. 1970. Extraction and determination of phytic acid in beans (Phaseolus vulgaris). Cereal Chem. 47:288. Martinez-Torres, C. and Layrisse, M. 1970. Effect of amino acids on iron absorption from a staple vegetable food. Blood. 35:669. Meiners, C.R., Derise, N.L., Lau, H.C., Crews, M.G., Ritchey, S.J., and Murphy, E.W. 1976. The content of nine mineral elements in raw and cooked mature dry legumes. J. Agric. Food Chem. 24:1126. Miller, G.F., Guadagni, D.G., and Kon, S. 1973. Vitamin retention in bean products: Cooked, canned and instant bean powders. J. Food Sci. 38:493. Morris, E.R. and Ellis, R. 1976. Isolation of monoferric phytate from wheat bran and its biological value as an iron source to the rat. J. Nutr. 106:753. Morris, E.R., Simpson, K.M., and Cook, J.D. 1980. Dephytinized vs. nondephytinized wheat bran and iron absorption in man. Am. J. Clin. Nutr. 33:941. Narasinga Rao, 8.5. 1981. Physiology of iron absorption and supplementation. Brit. Med. Bull. 37:25. Oberleas, 0., Muhrer, M.E., and O'Dell, B.L. 1966. Dietary metal- complexing agents and zinc availability to the rat. J. Nutr. 90:56. 96 Oberleas, D. 1971 The determination of phytate and inositol phosphates. In "Methods of Biochemical Analysis." 0. Click, ed. Interscience, New York, New York. Oberleas, D. 1973. Phytates. In "Toxicants Occurring Naturally In Foods.“ National Academy of Sciences, Washington, D.C. O'Dell, B.L. 1969. Effect of dietary components upon zinc availability. Am. J. Clin. Nutr. 22:1315. O'Dell, B.L. and Savage, J.E. 1960. Effect of phytic acid on zinc availability. Proc. Soc. Exp. Biol. Med. 103:304. O'Dell, B.L., Burpo, C.E., and Savage, J.E. 1972. Evaluation of zinc availability in foodstuffs of plant and animal origin. J. Nutr. 102:653. Patel, K.M., Bedford, C.L., and Youngs, C.W. 1980. Amino acid and mineral profile of air-classified navy bean flour fractions. Cereal Chem. 57:125. Prasad, A.S. 1978. Iron. In “Trace Elements and Iron In Human Metabolism." M.M. Wintrobe, ed. Plenum Publishing Co., New York, New York. Rackis, J.J. 1974. Biological and physiological factors in soybeans. J. Am. Oil Chem. Soc. 51:161A. Ranhotra, 0.5. 1972. Hydrolysis during breadmaking of phytic acid in wheat potein concentrate. J. Food Sci. 37:12. Reinhold, J.G. 1971. High phytate content of rural Iranian bread: A possible cause of human zinc deficiency. Am. J. Clin. Nutr. 24:1204. Reinhold, J.G., Ismail-Beigi, F., and Faradji, B. 1975. Fibre vs. phytate as determinant of the availability of calcium, zinc and iron from breadstuffs. Nutr. Rep. Int. 12:75. Roberts, A.H. and Yudkin, J. 1960. Dietary phytate as a possible cause of magnesium deficiency. Nature. 185:823. Robertson, J.B. and VanSoest, P.J. 1977. Dietary fiber estimation in concentrate foodstuffs. J. Animal Sci. 45 (Suppl. 1):254. Rockland, L.B., Miller, G.F., and Hahn, D.M. 1977. Thiamine, pyridoxine, niacin, and folacin in quick cooking beans. J. Food Sci. 42:25. Rockland, L.B. and Radke, T.M. 1981. Legume protein quality. Food Tech. 35 (3):79. Sathe, V. and Krishnamurthy, K. 1953. Phytic acid and absorption of iron. Ind. J. Med. Res. 41:453. 97 Sgarbieri, V.C., Garruti, R.S., Moraes, M.A.C., Hartman, L. 1978. Nutritional and sensory evaluation of mixtures of soybean (Glycine max L.) and comnon bean (Phaseolus vulgaris) for direct use as human food. J. Food Sci. 43:208. Sharpe, L.M., Peacock, W.C., Cooke, R., and Harris, R.S. 1950. The effect of phytate and other food factors on iron absorption. J. Nutr. 41:433. Sinha, S.K. 1977. Nutritive value of food legumes. In "Food Legumes: Distribution, Adaptability and Biology of Yield." S.K. Sinha, ed. Food and Agriculture Organization of the United Nations, Rome, Italy. Sloane-Stanley, G.H. 1961. Phytase. In “Biochemists"Handbook." C. Long, ed. Van Nostrand Co., Princeton, New Jersey. Stein, M. 1976. Natural toxicants in selected leguminous seeds with special reference to their metabolism and behaviour on cooking and processing. Qual. Plant Pl. Fds. Hum. Nutr. 26:227. Thompson, L.U., Rea, R.L., and Jenkins, D.J.A. 1983. Effect of heat processing on hemagglutinin activity in red kidney beans. J. Food Sci. 48:235. Tobin, G. and Carpenter, K.J. 1978. The nutritional value of the dry bean (Phaseolus vulgaris): A literature review. Nutr. Abstr. and Rev. 48:920. Tyler, 0.0. 1979. Water and mineral metabolism. In "Review of Physiological Chemistry." H.A. Harper, V.N. Rodwell and P.A. Mayes, eds. Lange Medical Publications, Los Altos, California. Underwood, E.J. 1977. Iron. In “Trace Elements In Human and Animal Nutrition." E.J. Underwood, ed. Academic Press, New York, New York. Vohra, P., Gray, G.A., and Kratzer, F.H. 1965. Phytic acid-metal complexes. Proc. Soc. Exp. Biol. Med. 120:447. Walker, K.A. 1974. Changes in phytic acid and phytase during early development of Phaseolus vulgaris L. Planta 116:91. Walker, W.M. and Hymowitz, T. 1972. Simple correlations between certain mineral and organic components of common beans, peanuts, and cowpeas. Commun. Soil Sci. Plant Anal. 3:505. Watt, B.K. and Merrill, A. 1963. U.S. Dep. Agric. Handb. No. 8. Agricultural Research Service, Washington, D.C. Welch, R.M. and VanCampen, D.R. 1975. Iron availability to rats from soybeans. J. Nutr. 105:253. 98 Wheeler, E.L. and Ferrel, R.E. 1971. A method for phytic acid determination in wheat and wheat fractions. Cereal Chem. 48:313. Young, V.R. and Janghorbani, M. 1981. Soy proteins in human diets in relation to bioavailability of iron and zinc: A brief overview. Cereal Chem. 58:12. APPENDIX Composition of Standard and Test Diets Estimated To Contain 6, 12, and 24 ppm Added Iron 99 Table 18. Composition of standard diets estimated to and 24 ppm added iron from ferrous sulfate. contain 6, 12, grams Ingredient 6 ppm 12 ppm 24 ppm Cerelose 2081.22 2081.04 2080.68 Casein 600.00 600.00 600.00 Corn oil 150.00 150.00 150.00 NaH2P04oH20 60.00 60.00 60.00 CaCO3 60.00 60.00 60.00 KCl 15.00 15.00 15.00 NaCl 15.00 15.00 15.00 Mineral premix 8.10 8.10 8.10 Choline chloride 4.50 4.50 4.50 Vitamin premix 3.00 3.00 3.00 FeSO4g7H20Inix 0.18 0.36 0.72 dl-Methionine 3.00 3.00 3.00 1All ingredients were obtained from the same sources as those in the basal diet (p. 45). Prepared with cerelose such that 1 g of mix contained 0.1 9 Fe. 100 Table 19. Composition of test diets estimated to contain 6, 12, and 24 ppm added iron from navy hull flour.1 grams Ingredient 6 ppm 12 ppm 24 ppm Cerelose 1911.47 1741.54 1401.68 Casein 551.48 502.97 405.93 Corn oil 150.00 150.00 150.00 NaH2P04'H20 60.00 60.00 60.00 CaCO3 60.00 60.00 60.00 KCl 15.00 15.00 15.00 NaCl 15.00 15.00 15.00 Mineral premix 8.10 8.10 8.10 Choline chloride 4.50 4.50 4.50 Vitamin premix 3.00 3.00 3.00 Bean flour 218.45 436.89 873.79 dl-Methionine 3.00 3.00 3.00 1All ingredients were obtained from the same sources as those in the basal diet (p. 45). 101 Table 20. Composition of test diets estimated to contain 6, 12, and 24 ppm added iron from navy starch II flour.1 grams Ingredient 6 ppm 12 ppm 24 ppm Cerelose 1856.43 1631.46 1181.51 Casein 558.70 517.40 434.80 Corn oil 150.00 150.00 150.00 NaH2P04°H20 60.00 60.00 60.00 CaC03 60.00 60.00 60.00 KCl 15.00 15.00 15.00 NaCl 15.00 15.00 15.00 Mineral premix 8.10 8.10 8.10 Choline chloride 4.50 4.50 4.50 Vitamin premix 3.00 3.00 3.00 Bean flour 266.27 532.54 1065.09 dl-Methionine 3.00 3.00 3.00 1All ingredients were obtained from the same sources as those in the basal diet (p. 45). 102 Table 21. Composition of test diets estimated to contfin 6, 12, and 24 ppm added iron from navy protein flour. grams Ingredient 6 ppm 12 ppm 24 ppm Cerelose 2007.19 1883.53 1784.59 Casein 541.56 444.15 366.23 Corn oil 150.00 150.00 150.00 NaH2P04-H20 60.00 60.00 60.00 CaCO3 60.00 60.00 60.00 KCl 15.00 15.00 15.00 NaCl 15.00 15.00 15.00 Mineral premix 8.10 8.10 8.10 Choline chloride 4.50 4.50 4.50 Vitamin premix 3.00 3.00 3.00 Bean flour 132.65 353.72 530.58 dl-Methionine 3.00 3.00 3.00 1All ingredients were basal diet (p. 45). obtained from the same sources as those in the