THE RIBOFLAVlN AND THIAMINE CONTENT OF DIFFERENT VARIETIES OF MICHIGAN BEANS, RAW AND COOKED Thesis for tho Degree 0% M. S MlCHtGAN. STATE COLLEGE Yu Jui Li 1949 0-169 This is to certify that the thesis entitled "The Riboflavin and Thiamine Content of Different Varieties of Michigan Beans, Raw and Cooked" presented hg Yu Jui Li has been accepted towards fulfillment of the requirements for Master of Science dmywein EQQQS and Nutrition 232w fl. W Majt )I' PrOlCSSUI' / Date A ‘: 0 4“; _ _— _-_ .IA _..__.-__ —-_A._ THE RIBOFLAVIN AND THIAMII‘IE COI-‘TENT OF DIFFFREMT VARIEPIES OF MICHIGAN B WIS , RAW AND COOKED BY Yu Jui Li .‘ A THESIS Submitted to the School of Graduate Studies of Michigan State College of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of MASTEi OF SCI BVCE Department of Foods and Nutrition School of Home Ebonomics 1949 THESIS ACKl'lCWLEDGT»TEllT The writer wishes to express her sincere gratitude to Dr. Dena Cederquist and Dr. Margaret A. Ohlson for their in- valuable guidance in making this study possible, to Dr. E. E. Dom for. his contribution of the testing material, to Miss Louise Kelley for providing samples of the baked beans, and to Miss Wilma Brewer and Mrs. Helen Tobey for their interest and encouragement . ***#***#** **¥#*#*# ****#t ##1## 11* * 31834.8 TABLE OF CONTENTS Page INTRODUCTION..."............................................ 1 REVIEW OF LITERATURE......................................... 3 Methods for the Determination of Thiamine......v......... 3 Methods for the Determination of Riboflavin............. 6 The Effect of Processing on the Thiamine and Riboflavin Content of Food.................................... 8 EKPERIMENTAL PROCEDURE....................................... 13 Baking of Beans......................................... l3 Moisture Determinations.................s............... 16 Vitamin Assay: Hydrolysis of Sample.................... 1'7 Vitamin Assay: Thiamine Analyses....................... 17 Vitamin Assay: Riboflavin Analyses..................... 18 RESULTS AND DISCUSSION....................................... 20 Thiamine and Riboflavin Content of Raw Beans............ 20 Correlation between Thiamine and Riboflavin Content of Raw ans.......................................... 23 Losses of Thiamineand Riboflavin in Baking Navy Beans.. 24 SUTxEMARY AND CONCLUSIONS".................................... 29 LITMTURE CITEDOOOOOOOODOOOOOIOOOOOOOOOOI...OOOOOOOOOOOOOOCO 31 APPEIDIXOOOOOO0.0.000...IOOOOOOOCOOOOOQOOO...OOOOIOOOOOOOOOOO 37 TABLES Number Title Page I Thiamine and Riboflavin Content 03 Beans Reported by Different Investigators......... 11 II Distribution of Navy Bean Samples Analyzed for Thiamine and Riboflavin Content............. 14 III RBCipe for the Baked 338.118.00.000eeeeeooeoeeeeeeo -15 IV Recoveries of Thiamine and Riboflavin on the Raw and Baked Bangoeeeoeoeeeoeoeeeeecoco... 19 V Thiamine and Riboflavin Content of One Variety and Five Strains of Michigan Navy Beans..... 21 VI Analysis of Variance of Thiamine Content in Raw BeanSOIOOOOOOOOOOOOOOOOOOOOO0.00.0.00... 22 VII Analysis of Variance of Riboflavin Content in Raw 338.113.000.00000.00.000.000...0.0.0000... 23 VIII Cooking Losses of Thiamine of Five Samples of BeaDSOOOOOCOOOOOOO0.0.0.000...0.00.00.00.00. 25 IX Cooking Losses of Riboflavin of Five Samples of BwnSOOOOOOOOO0.0.0.0000...OOOOOOOOOOOIOCOOC 26 X Thiamine and Riboflavin Content in Baked Samples ‘ of One Variety and Five Strains of Michigan Ilavy BeanSCOOOOIO...OOOOOOCOOOOIOOOOOOOOOOOO 27 XI Amount of Thiamine and Riboflavin Added to Each POt 0f Baked Beanssoo....................u. 28 Table I Table II Table III Table IV . APPENDIX Thiamine in Raw Navy BMSoooooooeeeooeeoo RibOflaVin in Raw Navy Ban-80000000000000. ThiMine in Baked Navy BMoeeooeooeoeeoo RibOleVin in Baked Navy Beans............ Page 37 58 39 40 INTRODUCTION Navy beans (Phaseolus Vulgaris) and other legumes generally have been considered as economical sources of body building protein and energy. Their use as food has increased during the past few years, therefore their overall nutritive value is considered to be of inter- est. It seems that seeds of plants are one of the richer sources of thiamine and riboflavin among the plant tissues in that they are prepared for the growing of young plants. In Michigan, the navy bean is one of the most important and dependable field crops. In 1915, the Robust bean, a variety of the white pea or navy type was introduced by F. A. Spragg of Michigan State College Agricultural Experimental Station. The Michelete bean was developed by E. E. Down.and J. W. Thayer Jr., in 1937 from a cross between the Robust and Early Prolific varieties. The Michelete variety possesses the desired characteristic of uniformity in size and shape and has greater resistance than other varieties to virus mosaic and other diseases. Studies by Fixsen (1938), Arnold and Eavehjem (1939) on the chemi- cal properties of thiamine and riboflavin showed that thiamine is rela- tively heat labile and that riboflavin is markedly heat stable. Extent of vitamin cooking losses depended upon the conditions under which the food was prepared. The process of cooking, the amount of cooking water added, time, temperature and many other details are all important in determining the rate of destruction of the vitamins. Since chemical and microbiological analytical methods have been developed, numerous determinations on thiamine and riboflavin of foods, raw and prepared, -1- were made for choosing of varieties having high contents and for study- ing the actual retention of the nutritive value after different methods .o of processing. The purpose of this experiment was to determine the difference in thiamine and riboflavin content of six varieties of Michigan beans grown under controlled conditions and to determine the effect of baking - on the thiamine and riboflavin content of these beans. -2- REVIEW OF LITERATURE Methods for the Determination of Thiamine Several biological methods for the determination of thiamine were used before the chenical methods were developed. The biological methods most generally used include the rat-growth method of Sherman and Chase (1931), the pigeon weight-maintenance method and the bradycardia method. In 1934, Schopfer proposed a quantitative test using a mold growth method. A technique was described for estimating thiamine by means of the growth of mold which does not normally grow in a synthetic medium. 0n the addition of pure crystalline thiamine, the mold is stimulated. to considerable growth. With increasing concentration of thiamine, the . growth of the organism is proportionately increased up to a maximum point. He believed that the organism used thiamine to synthesize its own growth factor. Sinclair (1938) showed that the above method was not specific. Schultz, Atkins and Frey (1937) used a fermentation method based on Sinclair's idea that the fate of alcoholic fermentation is power- . fully stimulated by the presence of thiamine in the fem-ntation mixture. The first chemical test was published by Kinnersly and Peters (1934). A diazotized compound of thiamine and sulfanilic acid in the presence of a buffer and formaldehyde was found. This compound which is pink colored was used for a qualitative test. In 1938, the same investigators intro- duced the use of takadiastase to liberate free thiamine. Prabluda and McCollum (1939) proposed the use of diazotized p-amino-acetophenone the intensity of the color being a measure of the vitamin concentration. Melnick and Field (1939) extended this method, released the phosphorylated thiamine by incubation with yeast phosphate, and found that permutit was the only adsorbent that would remove the vitamin and permit a 100 percent elution with dilute sulfuric acid, yielding a solution which could be rendered suitable for. analysis. Burnett, Peacock and Brown (1940) modified the Prabluda-LicCollum reaction by adsorbing the thia- mine on Superfiltrol. Kirch and Bergeim (1942) described a method in which the urinary thiamine was coupled with diazotized ethyl-p-amino- benzoate producing a pink to red color. The trichloroacetate of this amine was fairly stable in alcoholic solution. That the enzymatic di- gestion converted the cocarboxylase to free thiamine without any des- truction of the thiamine molecule was also reported. Teeri (1948) men- tioned that thiamine under certain conditions produces with cyanogen bromide a colored compound measurable at the wave length commonly employ- ed for the quantitative determination of nicotinic acid. The thiochrome method which is generally used was introduced in 1935 when Peters; Berger, Bergel and Todd found that a pure thiamine prepara- tion could be converted by oxidation in aqueous solution to a pale yellow sulfur containing compound, thiochrome, which showed an intense sky blue fluorescence. Jansen (1936) pointed out that thiamine could be measured by oxidation to thiochrome with alkaline ferricyanide. Recovery of thia- mine by this method was not quantitative. Hennessy and Cerecedo (1939) used an enzyme prepared from defatted beef kidney in acid solution for extraction, and subsequmtly adsorbed the vitamin on Hecalso. The fluorescence was measured photoelectrically. The result obtained by this method agreed well with those using biological tests. Harris and Wang (1939) removed the interfering: materials of urine samples by washing with an equal volume of isobutyl alcohol. The washed urine was oxi- dized to convert thiamine into thiochrome, extracted with isobutyl alcohol and cleared with anhydrous sodium sulfate. In the measurement of fluorescence, a standard solution of thiochrome was added drop by drop to a blank used as a control, whilerat the same time an equal vol- ume of isobutyl alcohol was added to the unknown.until the fluorescence of the two was matched. Pyke (1939) employed pepsin digestion followed by takadiastase to obtain a complete liberation of thiamine. Conner and Straub (1941) described the optimal conditions for the oxidation of thiamine to thio- chrome. The same authors and Halliday and Deuel (1941) reported that takadiastase and other related enzymes with high phosphate activity are capable of hydrolyzing cocarboxylase quantitatively into free thiamine and phosphatase. The presence of protein interferes'With the hydrolysis. The combined thiamine is in a complex form. Takadiastase was unable to ; separate the thiamine complex unless aided by a proteolytic enzyme.. Cheldelin, Eppright, Snell and Guirard (1942) suggested the use of both takadiastase and papain for the liberation of the free vitamin. Johannson and Rich (1941) appliedthe sthiochrane method to the analysis of cereal and cereal products. Andrews (1944) reported a col- lective study of thiamine concentration in cereals. Numerous microbiological methods for the determination of thiamine have>appeared in the literature in the last few years. These include the methods of Niven and Smiley (1943) and Sarett and Cheldelin (1944). -5- Since the thiochrome method was used in this study, the microbiological methods will not be reviewed. Methods for the Determinatipn _o_f_ Riboflavin Chick and Roscoe (1928), Sherman and his coworker, (1931-35) used the rat growth method for the determination of riboflavin and defined the Bourquin-Sherman rat unit as that amount of riboflavin which when fed as daily doses induced a gain of 3 gm. per week in a standardised. experimmtal animal. The animl is fed a basal ration which is suffi- ciently free from riboflavin to result in loss of weight during the test period. Recently the microbiological method of Snell and Strong (1939) was investigated by measuring the influence on both the cell growth and the acid production of Laotobacillus Caseiue grown on a synthetic medium free of riboflavin. Very satisfactory results were obtained. Kuhn and Wagner-Jauregg (1933) first noted the yellow green fluor- escence of riboflavin. These same authors and Kaltschmitt (1934) studied the riboflavin distribution in plants as lumiflavin by means of a photo- meter. Cohm (1935) measured the fluorescence of unknown solutions with a selenium cell method. Koschara (1935) used a method based on measure- ment of the light absorption of the vitamin extract rather than on its fluorescence. He also introduced the use of permanganate to oxidize interfering pigments and destroyed excess potassium permanganate with hydrogen pa'bxide. Hodson and Norris (1939) described a fluorescent method for use with foods. Riboflavin and the interfering pigments were reduced with a mixture of sodium hyposulfite and stannous chloride. The -6- reduced riboflavin was oacidized by bubbling air through the solution and its fluorescence measured by the use of a fluorophotometer. Sullivan and Norris (1939) measured the fluorescent reading of a sam- ple in a photometer. The sample was then reduced with sodium hyposul- fite. Reading of the reduced sample was also taken. The difference of the two photometric readings was regarded as the riboflavin value of the sample. Farrebee (1940) introduced the use of two Fuller's earth prepara- tions, Floridin and Supersorb, for the adsorption of riboflavin. The vitamin was eluted from these adsorbents with a solution of 20 percmt pyridine in two percent acetic acid. The eluate was treated with potas- sium permanganate and hydrogen peroxide prior to measuring the fluores- cence. Najjar (1941) applied the fluorometric method to urine and other biological fluids. The statement was made that two sources of error in the methods in which fluorescence was measured in aqueous solutions were turbidity of the solution and the formation of gaseous emulsions. The addition of potassium permanganate and hydrogen peroxide resulted in the formation of minute bubbles of oxygen which tended to ruin dispersed in the medium. This gave a fiwhitish" tint to the riboflavin fluores- cence and interfered with the accuracy of the fluorescence measurements. Najjar measured riboflavin in a non-aqueous medium by saturating the pyri- dine extract with anhydrous sodium sulfate. Conner and Straub (1941) modified the fluorometric method and combined it with the thiochrome method for the determination of the two vitamins, thiamine and riboflavin in food products. Andrews (1944) reported the collaborative study of riboflavin assay methods. Large variations in the results of individual -7- collaborators were found. That variation in the efficiency of different lots of Florisil were partially responsible for the difference were indi- cated. Rubin and De Ritter (1945) found that riboflavin was adsorbed more efficiently by Florisil from simple solution than extracts of high potency, but found that the use of Florisil may cause errors ranging up to 30 percent; the factors influencing the adsorption included the . clarity, volume and concentration of the extracts. Slater and Morell (1946) modified Najjar's method by using a larger amount of potassium permanganate and an internal standard and found good agreemmt between values obtained by this method and those from microbiological assays. De Ritter, Moore, Heischberg and Rubin (1948) stated that assays by a Florisil adsorption procedure includingpermanganate treatment of the eluate were slightly high as compared to other methods. In toms of ab- solute amounts of riboflavin, these differences were small, but at a very low riboflavin level, the percentage differences may be high. The validity of the use of a mixture of takadiastase and papain for the liberation of riboflavin from foods was considered to be the most effective ones by Cheldelin, et 9.1., (1942) and was confirmed by Rosner, Lerner and Cannon (1949). The Effect 23 Processing 93 the Thiamine and Riboflavin Contait 23 Foods Concerning the effect of processing of foods on their thiamine and riboflavin content, Fixsen (1938), Arnold and Elvehjan (1939) stated that thiamine shows considerable resistance to heat in an acid or slightly acid medium. Ecposure to temperatures of 100°-~120o C. resulted in greater losses than were observed at lower temperatures even in an acid medium, -8- while in an alkaline medium.there was extensive destruction at low'teu- peratures. Beadle, Greenwood and Kraybill (1943) reported that the des— truction of thiamine upon heating is a function of the temperature, time of heating, the pH of the medium.and the nature of other substances present. Fixsen (1938) and Mhnsell (1940) found that no loss of ribo- flavin resulted from.exposure to the temperature associated with cook- ing unless the medium was alkaline. Solubility in water will be the cause of losses in riboflavin when vegetables are boiled and the cock- ing water rejected. The study of Arnold and Envehjem (1939) reported a 60-80 percent less of thiamine due to heating. Miller (1945) studied the thiamine content of Japanese soy bean products. Seventy-nine per- cent loss of thiamine in cooking was found. Fenton, et al., (1945) reported that approximately two-thirds of the thiamine and riboflavin. were retained in peas during cooking. Greenwood (1938) reported that adding of soda is an effective method of softening the seed costs of beans. Lents (193B), Aughey and Daniel (1940), Johnson, Schauer and Daniel (1944) stated that soda may shorten the cooking time and has no effect on thiamine and riboflavin content. Several papers were published where a higher riboflavin content was Observed after cooking than was observed in the unprocessed mater- ials. These findings were not considered as errors in theranalytical procedures. Streightoff, et al., (1946) reported an average riboflavin retention totaling 122 percent for boiled carrots plus boiling water in ten batches of stored miscellaneous varieties and of 112 percent in ten batches of fresh Chantenay coreless carrots during large quantity preparation. Tucker, Hinman and Halliday (1946) stated that during braising, frying and broiling of beef there appeared to be no loss of riboflavin by any of the methods of cooking and in many cases there appeared to be a gain. The complex form in which riboflavin is formed or released during cooking is of such a nature that the vitamin can not be liberated by digesting or autoclaving under acid condition nor by enzyme hydrolysis. Watts, Peng and Esselbaugh (1948) made the statement that the enzymes within the meat itself bring about a progressive in- crease in the riboflavin liberated if opportunity is allowed for autodi- gestion in the presence of preservatives. This enzymatic liberation of riboflavin probably continues at the low temperatures of freezing storage and maybe greatly accelerated at higher temperatures in cooking. Few data relative to the thiamine and riboflavin content of mvy beans appeared in the literature. Table I presents a summary of the vitamin content of navy beans as reported by several investigators. The nutritive values of the cooked beans are also included. -10- Table I Thiamine and Riboflavin Content of Beans Reported by Different Investigators Name of Investigators Year Name of Beans @hiamine Content Riboflavin Content _, (Brady- Baker d'Wright 1935 Bean, Haricut 1.2 ‘cardia_ unit) Bean, Baked trace , s F‘ixsen a: Roscoe 1938 Been, Haricut 50-120 LIL/100 gm. Baked, & Canned trace _ Mhnsell 1940 Navy been 170 I.U./100 gm. Aughey, Daniel 1940 Navy bean 250 I.U./100 gm. Crooked 24o 1.11.1100 gm. 'Kelley, Dietrich 1940 Michelete 170 I.U./100 gm. & Porter 1941 Baked in soak- ing water 230 1.g./1oo gm. munsell 1942 Navy bean, 300,325 meg./100 gm. dried ss Booher, Hartzler 1942 Michelete 600 mcg/lOO gm. & Hewston Robust 720 " Navy 750 " Ives, Wagner 1945 Baked beans 210 mcg./100 gm. 54o mcg./lOO gm. Elvehjem & Strong new English type Daniel 1.: Norris 1945 Robust 630 mcg./100 gm. 179 mcg./100 gm. Michelete 656 meg./100 gm. 170 meg.[100_gm, * The international unit is defined as the potency of 3 of pure thiamine hydrochloride. ‘ ts Mcg. is used as an abbreviation of microgram. -11.. micrograms Among the reported studies, the work of Kelley, Dietrich and Porter (1940, 1941) and Daniel and Norris (1945) were of particular interest. Kelley, Dietrich and Porter used the rat growth method to determine the thiaminevalue of eight varieties of beans grown in two localities in Michigan. Results obtained indicated that the Blue pod and Robust varie- ties were highest in thiamine content and Michelete was of medium value. The effect of cooking on the thiamine content of Michelete and Cranberry beans was studied using the same method. Thiamine content of all sam- ples of beans cooked by the different methods was found to be higher than that of the original raw beans. The assumption was made that the cooking processes rendered the thiamine content of the beans more readily available to the animals than was the thiamine in the uncooked beans. Daniel and Norris studied the riboflavin, niacin and thiamine con- tents of dried legumes. The thiochrome method and fermentation method were used for the thiamine analysis. The fluorometric method and micro- biological method were used for the riboflavin analysis. Eccellent agree- ment was found between the two methods that were used in the assay of each vitamin in one variety of legume. Vitamin contents of Michelete and Robust beans were reported in Table I. -12- EXP ERIMEEWAL PROC EDURE One variety and strains of five different crosses of Michigan navy beans were obtained from the Farm Crop Department in Michigan State College. In the cooking trials, Michelete was used as a standard for comparison of palatability. Other beans represent the newly crossed seeds. The distribution of the samples is show in Table II. A total of 46 raw and the same number of baked beans were analyzed for thiamine and riboflavin content. According to the reports of Loofbourow and Harris (1942), William and Chedelin (1942) and Def-Torre and Brom (1944) riboflavin can be des- troyed by visible light. Therefore all experiments in this study were carried in semidarknes s. For the determination of thiamine, the thiochrome method of Hennessy and Cerecedo (1939) as developed by Michelson, Condiff and Keys (1945) for the analysis of thiamine in urine was used. The chenical method of Hodson and Norris (1939) with modification by Conner and Straub (1941) and Keys (1944) was used for the estimation of riboflavin. Fluorescence of the vitamins was measured with a fluorescence meter."I Baking _o__f_ Beans: The baked beans were prepared by Miss Louise Kelley as a part of an experimmt station project on the palatability of different samples of beans. The recipe for the baked beans is given in Table III. * A Coleman photofluorometer and a Lumetron photofluorometer were used. -13- Table II Distribution of Navy Bean Samples Analyzed for Thiamineuand Riboflavin.Content Variety Strains of Different Crosses ‘Michelete Robust x Robust x Robust x Robust x Robust x Early Prolific Crawford Darling Mexican.Tree Hunter Sample No. Sample No. Sample No. Sample No. Sample No. Sample No. 801 802 803 822 866 836 811 812 804 823 872 842 821 813 805 824 873 843 831 814 806 825 841 815 844 826 851 816 845 832 861 846 833 871 852 .834 33 B53 835 854 855 856 862 863 864 865 -14- Table III Recipe For The Baked Beans Approximate 'Weight Ingredient measure (gm.) Source Dried beans 1.5 cups 310.0 variable Mblasses 3.0 tablespoons 70.0 Grandma's Old Fashioned Mustard 1.75 teaspoons 1.3 Bulk Salt 0.75 teaspoons 5.0 Iodized Salt pork 0.20 pound 70.0 Soda 0.15 teaspoons not weighed Bicarbonate ; -1 5.. Six samples of beans were baked each time, one of the six being the Michelete variety. Before baking, the selected dried beans were washed through two waters, drained and rinsed with distilled water. Three hundred and ten grams of beans were soaked in 600 cc. of distilled water overnight in a Pyrex bowl. In the morning, the beans and the soak- ing water were transferred to an aluminum pan and were allowed to boil for five minutes with a small amount of baking soda added to it. The soda water was then drained off and the beans were washed with distilled water. Slices of salt pork were put at the bottom of a bean pot. The beans were transferred into the bean pot. Molasses, salt, mustard were added and the beans covered with boiling. distilled water, baked in a preheated oven at 2500 F. for seven hours. The cover of the pot was re- moved during the last hour of baking. Five samples of the beans were used to compare the cooking differ- ances in the riboflavin and thiamine value due to the ingredients added to the baked beans. mch kind of beans were baked in two pots. One sam- ple was cooked with all the ingredients among which pork and molasses were emtranely interesting. Another sample was cooked under the same condition without any ingredient concept the soda. Analyses of the vita- mins were made using the same methods as described. Moisture Determinati org Samples of raw beans and freshly baked beans were put in weighed moisture dishes and were dried in an oven between 70°-75° C. for 48 hours. _ Vitamin Assay: Hydrolysis of; Sample In hydrolyzing the samples, Hennessy's method (1941) was used. Fifty grams of freshly baked beans were ground in a Waring blender for 3 minutes with 100 cc. of 0.2 N sulfuric acid. One hundred grams of the slurry was put in an amber glass flask and hydrolyzed for one hour on a steam bath. After cooling, the pH was adjusted to 4.5 with 15 percent sodium hydroxide and branccresol green was used as an outside indicator. Ten millimeters of an acetic acid-sodium acetate buffer solution containing’0.l gm. of takadiastase and 0.1 gm. of papain were added to the sample. The flask was incubated over night at 45° C. The following morning, the samples were heated on a steam bath for 5-10 minutes to inactivate the enzymes; the sample was cooled and filtered through No. 12 Whatman pleated filter paper. The filtrate was used for assay of thiamine and riboflavin. For the analyses of the uncooked dried beans a twenty-five gram sam- ple was used and 200 m1. of 0.2‘ N sulfuric acid were added and than blend- ed as described above. The samples were transferred quantitatively to a 250 m1. volumetric flask after hydrolyzing with enzymes and then filtered. The vitamin content of pork, molasses and the enzymes were analyzed separately. The enzymes were found to contain insignificant amounts of riboflavin and thiamine. Vitamin Assay: Thiamine analyses Five milliliters of the filtrate were allowed to pass through a colunm of activated Decalso and drained. The column was washed with three portions of buffered water. The adsorbed thiamine was eluted with about 20 m1. of hot 25 percent KCl in 0.1 N HCl solution. The eluate was collected in a 25 m1. graduated cylinder and made to volume. Five milliliters of the KCl-HCI eluate was oxidized in an oxidizing chamber which contained three ml. of 15 percent sodium hydroxide and 0.1 ml. of freshly prepared one percent potassium ferricyanide. Fifteen milliliters of redistilled isobutyl alcohol were added and the chamber was shaken for 1:33— minutes, centrifuged for 1%; minutes, and the water layer removed with a suction pipette. The isobutyl alcohol layer was cleared with ap- proximately 3 gms. of anhydrous sodium sulfate. The extract was trans- ferred to a glass cuvette and its fluorescence was measured in the fluoro- photometer which was standardized against a thiamine solution containing 0.5 mcg. of thiamine per m1. Quinine sulfate solution was used as a sec- ondary standard because it is stable. Readings of the blank of each sam- ple were measured in the same way without the ferricyanide solution. Andrews (1944) recommended that the highest fluorescence values were ob- tained when the alkali was added first and followed immediately by the ferricyanide and after 1%.;- minutes of shaking. Vitamin Assay: Riboflavin Analyses Ten milliliters of the filtrate from hydrolysate of the raw or cook- ed beans were allowed to pass through a column of activated Florisil. The columns were rinsed with two successive 10 ml. portions of hot dis- tilled water. Portions of hot pyridine-acetic acid solution of total volume 35 cc. were passed through and collected in a 50 m1. volumetric flask. One milliliter of two percent potassium permanganate solution was added and followed by a shaking of three minutes. One milliliter of three percent hydrogen peroxide was used to decolorize the excess permanganate. The sample was made to volume and read in the fluorophoto- meter. The fluorescence of samples was read against a standard solution containing 0.1-0.15 mcg. of riboflavin per m1. A reagent blank was pre- pared for each set of readings. Recoveries of thiamine and riboflavin were measured by adding stand- ard solutions to the slurries of both the raw and the baked beans. Re- sults are given in Table IV. Table IV Recoveries of Thiamine and Riboflavin on the Raw and Baked Beans Sample Thiamine Riboflavin Amount in Amount Recovery Amount in Amount Recovery sample added sample added Mcg./gm.1 mcg. % mcg./gm.:l mcg. % Raw bean 2.65 1.00 98.89 1.72 1.60 96.75 Baked bean 10.25 2.00 98.54 1.55 1.60 99.21 1 Dry basis —19- RESULTS AND DISCUSSION Thiamine and Riboflavin Content _o_f_ Raw Beans Results of the study of the thiamine and riboflavin content of raw beans are presented in.Tab1e V. The thiamdne values are all higher than those reported by Kelley, Dietrich and Porter (1940), of Booher, et al., (1942) and of Daniel and Norris (1945). (See Table I). The one variety and the five strains as arranged in descending order in relation to the thiamine content are Robust x Darling, Robust x maxican Tree, nichelete, Robust x Early Prolific, Robust at Hunter and Robust 3: Crawford. The mean values were 11.89 t 0.45 (standard deviation), 10.90 I 0.41, 10.84‘2 0.37, 10.053 0.55, 10.022 0.11 and 9.67: 1.54 micrograms per gram or dried beans respectively. The range of the thiamine values was from.7.28 to 12.97 mcg. per gm. of dried beans. The range of the riboflavin values was fran 1.35 to 1.95 mcg. per gm. of dried beans. These values are generally lower than those report- ed in the literature, (See Table I). Some of the values agree well with those of Daniel and Norris (1945). The one variety and the five strains as arranged in relation to the riboflavin content are Robust x Darling, Robust x Mexican Tree, Michelete, Robust x Hunter, Robust x Early Prolific and Robust x Crawford. The mean values were 1.81: 0.01, 1.721 0.01, 1.68:0.02, 1.66: 0.01, 1.63: 0.03, 1.58: 0.05 mcg. per gm. of dried beans respectively. According to the results of this comparison, the raw samples of'the Robust x Darling strain is considered to be the best source of thiamine and riboflavin in the series. The samples of Robust x Darling strain -20- Thiamine and Riboflavin Content of One Variety and Five Strains of Table V 1 Dry basis. -2 1.. dichigan Navy Beans Thiamine Riboflavin Variety Mean and mean and Standard Deviation Range Standard Deviation Range 1 1 1 1 ‘Mcg./gm. meg/gm. mcg.(gm. mcg./gm. Nflchelete lO.B4-O.37 9.97-11.77 1.68-0.02 1.53-1.84 ‘Robust x g + Early Prolific . lO.05-O.55A_ 9.61-11.46 1.63-0.03 1.35-1.86 Robust x + + "Crawford 9.67-1.54 7.28-11.62 1.58-0;02 1.35-1.78 Robust x + + Darling 11.89-0.45 11.06-12.97 1.81-0.01 1.63-1.95 Robust X + .1. 156110311 Tree 10.90-0041 10.16-11.34 1.72-0.01 1.63-1.83 Robust x 4 Hunter 10.02i0.11 9.79-10.40 1.66-0.01 1.59-1.78 have the highest value in both thiamine and riboflavin content while the Robust x Crawford beans have the lowest value in both vitamins. In order to separate the variation between strains from the variation due to the replication of samples within each strain of beans, analysis of variance was used. The analysis for the thiamine values is shown in Table “‘0 Table VI Analysis of Variance of Thiamine Content in Raw Beans Source of Degree of Sum of Mean Value of F Variation Freedom Square Square Total 45 65.07 Between variety 5 31.53 6.31 7052 #‘ Within variety 40 33.54 0.84 Predicted valpifii (From Snedecor) Probability < .05 2.45 Probability < .01 3.51 From the above calculation of F value, the difference in thiamine value of samples between varieties is highly significant. The data of the riboflavin content of raw beans were also analyzed by using analysis of variance. Result is given in Table VII. -22 - Table VII Analysis of Variance of Riboflavin Content in Raw Beans Source of Degree of Sum.cf Mean Value of F Variation Freedom Square Square Total 45 1.148 Between variety 5 0.335 0.067 3.30 * Within variet4 40 0.813 0.203 Predicted value of F Probability 1: .05 2.45 Probability <: .01 3.51 There was a significant difference in the riboflavin content of different varieties. Correlation between Thiamine and Riboflavin Content of Raw Beans ”huh-.- _w-—-*- Correlation coefficient between the riboflavin and thiamine content of raw beans was calculated by using the equation: 8):}! - Sx SZ 11 I I“? ' 2 3x - y(s-Ll (a W<>> n n where x represents the thiamine content, y represents the riboflavin content. The correlation coefficient obtained was 0.44. The probability of significance of this value is given below. -23 - =Xglue of r (Fisher) Pmbability < .05 0.3044 Probability < .01 0.5932 The correlation coefficient between thiamine and riboflavin value of raw beans is highly significant. Losses .93 Thiamine- and Riboflavin in Baking Navy Beans The cooking losses in thiamine and riboflavin content were determined for five samples which were baked under the same conditions with and with- out molasses and salt pork. The differences in thiamine and riboflavin content between raw beans and those baked without added ingredients were used as a measure of the cooking losses. Results are shown in Tables VIII and IX. A decrease in both vitamins was shown. The average percent- age loss of thiamine is 70.88 percent, and the average loss of riboflavin is 21.09 percent. Thus it is shown that in the baking process the beans lose more thiamine than riboflavin. The thiamine and riboflavin content of baked beans are presented in Table X. The average, range and standard deviation arereported for each kind. Less varietal difference in the thiamine and riboflavin content of the baked samples appeared than in the samesamples of the raw beans. In the recipe used for the baked beans, a considerable amount of salt pork and molasses were added to each been pot. The riboflavin content of these ingredients is high enough to affect the result of the baked beans. Table XI shows the amount of vitamins added to each bean pot. 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Some investigators mentioned before, Streightoff, et al.. (1946) and Tuck, Hinman, and Halliday (1946) had found high retention of riboflavin after cooking of vegetables and.msat. These authors suggested that there was greater liberation of riboflavin in the cooked samples than that in the raw ones. This may be one of the reasons in this study that a high value of ribo- flavin was obtained after baking. The addition of salt pork and molasses may be of importance in raising the riboflavin value of the cooked camp ples, because a decrease in both vitamin values was obtained when the beans were baked with no added ingredients. The amount of thiamine in the baked beans cooked with ingredients is approximately 141 mcg. for each serving and the riboflavin is approximately 107 mcg. for each serving. Actual results of thiamine and riboflavin analysis of each sample of beans raw and.baked are tabulated in the Appendix Tables I, II, III and IV. -23- SUTTLARY AND CONCLUSIONS The differences in thiamine and riboflavin content of 46 raw and cooked samples representing one variety and strains of five crosses of Michigan navy beans were determined. The method of Mickelson, Condiff and Keys (1945) was used to determine the thiamine content of the beans; the riboflavin content was determined by the method of Connor and Straub (1941), modified by Keys (1944). . Analysis of variance of the thiamine and riboflavin contents of the raw beans of one variety and strains of five crosses showed that there was a highly significant difference in the thiamine content between the various lots of beans and that there was a significant difference in the riboflavin content between lots. The average thiamine contents were 11.89 mcg. per gm. of dry beans for Robust x Darling, 10.90 mcg. for Robust x Mexican Tree, 10.841mcg. for Michelete, 10.50 mcg. for Robust 3: Early Prolific, 10.02 mcg. for Robust 1 Hunter and 9.67 mcg. for Robust x Crawford, arranged in.des- sending order. The average riboflavin contents were 1.81 mcg. per gm. of dry beans for Robust x Darling, 1.72for Robust x Mexican Tree, 1.68 for Michelete, 1.66 for Robust x Hunter, 1.63 for Robust x Early Pro- lific and 1.58 for Robust 1 Crawford. The correlation coefficient between the thiamine and riboflavin content of the dried seeds was found to be highly significant. Five samples of the beans were baked without the addition of salt pork, and molasses, thiamine cooking losses averaged 70.88 percent and the cooking losses of riboflavin averaged 21.09 percent for the same samples. In the baking trials, when the beans were baked with added ingred- ients, a marked decrease in the thiamine content and a slight increase in the riboflavin content were observed. The increase in riboflavin content resulted from the addition of the salt pork and molasses. There appeared to be less difference in the thiamine content of different lots of baked beans than was observed in the same samples before baking. A 150 grams serving of thenbaked beans reported in this study con- tained approximately 141 mcg. of thiamine and 107 mcg. of riboflavin. -30- LITERATURE CITED Andrews, J. S. 1944 Report of the 1943-1944 Methods of Analysis Subcommittee on Thiamine Assay. Cereal Chem., 21: 388-397. Andrews, J. S. 1944 Report of the 1943-1944 Methods of Analysis Subcommittee on Riboflavin Assay. Cereal Chem., 21: 398-407. Andrews, J. S. and R. Nordgren 1941 The application of the Thic- chrome Method to the Thiamine.Ana1ysis of Cereals and Cereal Products. Cereal Chem., 18: 686-695. Arnold, A. and C. A. Eflvehjem 1939 Processing and Thiamine. Food Research, 4: 547-553. Aughey, E. and E. P. Daniel 1940 ETfect of Cooking upon the Thia- mine Ccntent of Foods. J. Nutr., 19: 285-296. Baker, B. Z. and ME D.‘Wright 1935 The Vitamin B1 Content of Foods. BiOCheme Jo, 29; 1802-18070 Barker, G., EL Bergel, and A. R. Todd 1935 Ber. deutsch. chem. Gesellsch. 68: 2257-2262. (Original not available for exami- nation; abstracted in Nutr. Abstr. Rev., 5: 943). Beadle, B. W., D. A. Greenwood and H. R. Kraybill 1943 Stability of Thiamine to Heat. J. Biol. Chem., 149: 339-347. Booher, L. EL and E. R. Hartzler 1939 The Vitamin Bl Content of Foods in Terms of Crystalline Thiamine. U. 8. Dept. Agri. Tech. Bu11., No. 707. Booher, L. E., E. R. Hartzler and E. M. Hewston 1942 A Compilation of the Vitamin Value of Foods in Relation to Processing and Other Variants. U. S. Dept. Agri. Ciro. Bull. No. 638.’ Cheldelin,'V. H., M. A. Eppright, E. E. Snell and B. M. Guirard 1942 Enzymatic Liberation of B Vitamins from.Plant and Animal Tissues. The University of Texas Publication, No. 4237: 15-36. Cheldelin,'V. H. and R. J. Williams 1942 The B Vitamin Content of Foods. The University of Texas Publication, No. 4237: 105-124. Chick, H. and M. H. Roscoe 1928 The Effect upon Young Rats of Vita- min B2 Deficiency and a Method for the Biological Assay of Vita- min Bz. Biochem. J., 22: 790-798. -31. Cohen, F. H. 1935 A Simple.Apparatus for Objective Fluorescence meas- urement by means of a Selenium.Cell. Rec. Trav. Chim. Pays-Bas, 1935 54: 133 (Original not available for examination; abstracted in thr. Abstr. Rev., 5 : 949). Conner, R. T. and G. J. Straub 1941 Determination of Thiamine by the ThiOChrGIne 111eth0de Ind. mg. Chemo Anal. we, 15: 380'384. Conner, R. T. and G. J. Straub 1941 Combined Determination of Ribo- flavin and Thiamine in Food Products. Ind. Eng. Chem. Anal. Ed., 13: _385-388. Daniel, L. and L. C. Norris 1945 The Riboflavin, Niacin and Thiamine Content of Dried Leguminous Seeds. J. Nutr., 30: 31-36. De Meere L. J. and'W. S. Brown 1944 Effect of Various Lighting Con- ditions on Riboflavin Solutions. Arch. Biochem., 5: 181-190. De Ritter, E., M. E. Moore, E. Hirschberg and S. H. Rubin 1948 Criti- que of Methods for the Determination of Riboflavin in Urine. J. Biol. Chem., 175: 883-892. Down, E. E. and J. W. Thayer Jr. 1937 The Michelete Bean. Rich. Agri. Expt. Sta. Special Bull., No. 295. Emmett,.A. D., G. Peacock and R. A. Brown 1940 Chemical Determination of Thiamine by a modification of the Melnick-Field Method. J. Biol. Chem., 135: 131-138. Farrebee, J. W. 1940 The Urinary Excretion of Riboflavin.F1uorometric Methods for its Estimation. J. Clin. Invest., 19: 251-260. Fenton, F., E. Gleim, M..Albury, K. Visnyl and J. R. MCCartney 1945 Effect of Large Quantity Preparation Procedures on Vitamin Reten- tion: Canned Peas. J. Am. Diet. Assoc. 21: 700-702. Fisher, R. A. 1934 Statistical methods for Research Wbrkers. 5th Ed. Fixsen, M..A. B. 1938 The Vitamin Content of Human Foods as affected by Processes of Cooking and Canning. (With Tables) Nutr. Abstr. Rev., 8: 281-307. Fixsen, M; A. B. and M. H. Roscoe 1938 Tables of the Vitamin.Content of Human and Animal Foods. Nutr. Abstr. Rev., 7: 823-867. Greenwood, M. L. 1938 The Vitamin B Content of Raw Pinto Beans. New Mex. Agr. Expt. Stat. Bull. No. 232. -32- Halliday, N. and H. J. Deuel Jr. 1941 The Presence of Free and Come bined Thiamine in Milk. J. Biol. Chem., 140: 555-561. Harris L. J. and Y. L. wang 1939 Methods for Assessing the Level of Nutrition of Human Subject. Estimation of Vitamin B1 in Urine by the Thiochrome Test. Biochem. J., 33: 1356-1369. Hennessy, D. J. 1941 Chemical Methods for the Determination of Vita- min B. Ind. Eng. Chema.Ana10 E30, 133 216-2180 Hennessy, D. J. and L. R. Cerecedo 1939 The Determination of Free and Phosphorylated Thiamine by a Modified Thiochrome Assay. J. Am. Hinman, W; F0, TuCker, R. E., L. M. Jana and E. G. Halliday 1946 EX- cessively High Riboflavin Retention During Braising of Beef. Ind. mg. Chm. Anal. R10, 18: 297-501. Hodson, A. Z. and L. C. Norris 1939 A Fluorometric Method for Deter- mining the Riboflavin Content of Foodstuffs. J. Biol. Chem., 131: 620-630. Ives, H., J. R. Eagner, C. A. Elvehjem.and F. M. Strong 1944 The Nutri- tive Value of Canned Foods. J. Nutr., 28: 117-121. Jansen, B. C. P. 1936 .A Chemical Determination of Aneurin (Vitamin B1) by the Thiochrome Reaction. Rec. Trav. Chim. Pays-Baa 55: 1046. (Original not available for examination; Abstracted in Nutr. Abstr. ReVo, 78 56). Johannson, H. and E. 0. Rich 1941 A modified Thiochrome Method for the Estimation of Vitamin B1 in'Wheat and Its Products. Cereal Chem.,18, 47.3-49.2.- - Johnson, C. H., L. Schauer, S. Rapaport and H. J. Deuel Jr. 1943 The Effect of Cooking with and without Sodium.Bicarbonate on the Thiamine, Riboflavin and Ascorbis Acid Content of Peas. J. Nutr., 26: 227-2390 Kelly, E., K. S. Dietrich and T. Porter 1940 Vitamin B1 Content of Eight Varieties of Beans Grown in Two Localities in Michigan. Food Research, 5: 253-262. Kelly, E. and T. Porter 1941 Effect of Cooking Upon the Vitamin B Content of Two Types of Beans Grown in Michigan. Food Researc , 6: 85-93. Keys, A. 1944 By Communication. Kinnersly, H. W. and R. A. Peter 1934 The Formaldehyde-azo-test for Vitamin 81' Biochem. J., 28: 667-670. Kinnersly, H. W. and R. A. Peters 1938 Note Upon the Preparation of Crude Cocarboxylase from Vitamin Bl by Yeast. Biochem. J. 32: 697-698. Kirch, E. R. and 0. Bergeim. 1942 The Chemical Determination of Thia- mine. J. Biol. Chem., 143: 575-588. Koschara, W. 1935 Urine Lyochromes. Hoppe-Seylor's Ztschr., 232: 101- 106. (Original not available for examination; abstracted in Nutr. Abstr. Rev., 5: 81). Kuhn, R. T. Wagner-Jauregg and H. Kaltshmitt 1934 Distribution of Flavins in Plants. Ber. d. deutsch. chem. Gesellsch. 67: 1452- 1451. (Original not available for examination; abstracted in Nutro ArStro Revs; 43 509). Kuhn, R. and T. Wagner-Jauregg 1933 Flavin Isolate from Egg White and Milk. Ber. d. deutsch. chem. Gesellsch. 66: 1577-1582. (Original not available for examination; abstracted in Nutr. Abstr. Rev., 3. 992). ' Lantz, EL M. 1938 The Effect of Different Methods of Cooking on the Vitamin B Content of Pinto Beans. New Mex. Agr. Ekpt. Sta. Bull. N0. 2540 Lantz, E. M. 1940 The Riboflavin and Vitamin BB Content of Pinto Beans and the Effect of Cooking on These Factors. J. Home Ebon., 32: 101-102. Loofbourow, J. R. and R. S. Harris 1942 The Evaluation of Fluorophoto- meter to be used in the Thiochrome Assay for Vitamin Bl' Cereal Melnick, D. and H. Field 1939 Chemical Determination of Vitamin 81' I. Reaction Between Thiamine and Diazotized p-amino-acetophenone. J. 8101. Chan‘s, 1278 505-5140 Melnick, D. and H.Fie1d 1939 Chemical Determination of Vitamin B . II. Method for EBtimation of the Thiamine Content of Biological Materials. J. BiOlo Chem., 1272 515-5300 Melnick, D. and H. Field 1939 Chemical Determination of Vitamin 81' III. Quantitative Enzymic Conversion of Cocarboxylase to the Free Vitamin. J. Biol. Chem., 127: 531-540. Mickelson, 0., H. Condiff and A. Keys 1945 The Determination of Thia- mine in Urine by Means of the Thiochrome Technique. J. Biol. Chem., 160: 361-370. ‘ Miller, C. D. 1945 Thiamine Content of-Japanese Soybean Products. J. m. Diet. ASSOCO, 218 430-432. Mhnsell, H. EL 1940 Vitamins and Their Occurance in Foods. Milbank Memorial Fund Quarterly, 18: 311-344. Bhnsell, H. E. 1942 Riboflavin Content of Some Common Foods. Food Research, 7: 85-95. Najjarg, V..A. 1941 The Fluorometric Determination of Riboflavin in Urine and Other Biological Fluids. J. Biol. Chem., 141: 355-364. Niven, C. F. Jr. and K. L. Smiley 1943 A Microbiological Assay Method for Thiamine. J. Biol. Chem., 150: 1-9. Peters, R. A. 1935 Vitamin B1 and Blue Fluorescent Compound. Nature, 1353 167. Prebluda, H. J. and E. V. McCollum 1939 A Chemical Reagent for Thia- mineo' J. 8101. Chane, 127: 495-5030 Rosner, L., E. Lerner and H. J. Cannon 1945 ‘Use of Enzyme in Riboflavin Determination. Inc. Eng. Chem. Anal. Ed., 17: 778-779. Rubin, S. H. and EL DeRitter 1945 The Adsorption of Riboflavin in Flori- 81‘10 Jo 31.01. Chan" 1588 639-6450 Sarett, H. P. and V. H. Cheldelin 1944 The Use of Lactobacillus Fermen- tum 36 for Thiamine.Assay. J. Biol. Chem., 155: 153-160. Schopfer, W. H. 1934 Plant Test for Vitamin B Z. Vitaminforsuch 4: 67-75. (Original not available for examination; abstracted in Nutr. Abstr. Rev., 5: 69). Schultz, A. 8., L. Atkin.and C. N. Frey 1937 A.Fermentation Test for Vitamin B. J. Am. Chem. Soc., 59: 2457-2460. Sherman, H. C. and A. Bourquin' 1931 Quantitative Determination of Vita- Min G(Bz)o Jo A1“. Chan. 8000’ 538 3501-5505. Sherman, H. C. and E. F. Chase 1931 A quantitative Study of the Deter- mination of the Antiaeuritic Vitamin B. J. Am. Chem. Soc., 31: 3506-3510. -35- Sinclair, H. M. 1938 The Estimation of Vitamin B1 in Blood. Biochem. J., 32: 2185—2199. Slater, EL C. and D. B. Morell 1946 A MOdification of the F1uoro-' metric Method of Determining Riboflavin in Biological Materials. Biochem. J., 402 644-6520 Snedecor, G. W. 1946 Statiscal Methods 4th Ed. Snell, E. E. and F. M. Strong 1939 The Effect of Riboflavin and Cert- tain Synthetic Flavins on the Growth of Lactic Acid Bacteria. En- zymologia, 6: 186-193. \ a Streightoff, F., H. E. Munsell, B. Ben-Der, M. L..0rr, M. H., Leonard, S. R. Etekiel and F. G. Koch 1946 Effect of Large-scale Methods of Preparation on the Vitamin Content of Food: II. Carrots. J. Am. Diet. Assoc., 22: 511-518. Sullivan, R. and L. C. Norris 1939 Determining Riboflavin in Dried Til-11k HOdUCtS. Ind. Dig. Chan. Ana-1. mo, 11: 535-540. Teeri, A. E. 1948 Thiamine and the Cyanogen Bromide Reaction. J. 3101. Chane, 173: 503-5050 Tucker, R. E., W. F. Hinman and E. G. Halliday 1946 The Reactions of Thiamine and Riboflavin in Beef Cut During Braising, Frying and Broiling. J. Am. Diet. Assoc., 22: 877-881. 'Watts, B. H., D. H. Peng and N. C. EBselbaugh 1948 The Enzymatic Er- traction of Riboflavin from Pork for the Fluorometric Determina- tion. Jo B101. 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