EVALUATION OF POTATO PROTEIN , BY AMINO ACID ANALYSIS AND WE BINDING Thesis for the Degree of Ph. D.« MICHIGAN STATE UNIVERSITY; MIKLOS SANDOR KALDY 9'” LIBRARY ‘1 Michigan State Umversity This is to certify that the thesis entitled EVALUATION OF POTATO PROTEIN BY AMINO ACID ANALYSIS AND DYE-BINDING presented by MIKLOS SANDOR KALDY has been accepted towards fulfillment of the requirements for Ph.D. degreein Food Science I - ’ . O .1/ I /) 7/” / l/ I lé) ’5' Date April 21, 1971 0-7639 BINDING BY A . IIDAG & SIINS' am «my mc. ' ‘ .DERS .' ABSTRACT EVALUATION OF POTATO PROTEIN BY AMINO ACID ANALYSIS AND DYE-BINDING BY Miklos Sandor Kaldy The quality of proteins in six varieties of potatoes (Solanum tuberosum L.) was evaluated by amino acid analysis. Seventeen amino acids were determined by means of a Beckman Model 120C Amino Acid Analyzer. Methionine and cystine + cysteine were oxidized with per- formic acid to methionine sulfone and cysteic acid, re- spectively, prior to acid hydrolysis. TryptOphan was determined colorimetrically after hydrolysis with pronase. Protein scores for the varieties Russet Burbank, #58, #321-65, #322-6, #709 and #711-3 were 73, 78, 60, 62, 73 and 68, respectively, with an average of 69. Methionine was the limiting amino acid in these potato varieties. Fifteen free amino acids of the same varieties of potatoes were determined by the Technicon Amino Acid Analyzer. The variability among varieties in free amino acid content was greater than that in total amino acid content. Proportionally, there was more methionine among Miklos Sandor Kaldy the free amino acids than among the total amino acids. On the average, 11% of the total N of potatoes was present in the form of free amino acids, 69% in the form of bound amino acids and 20% was unaccounted. Two colorimetric methods were developed for-the rapid estimation of "total protein" in potatoes. Color development with ninhydrin for freeze-dried potatoes and orange G dye-binding for raw potatoes provided good cor- relations with the "total protein" content (% N x 6.25) of this commodity. The correlation coefficient and the standard error of estimate was + 0.9987 and 0.09%, re- spectively, for the ninhydrin method and + 0.9827 and 0.07%, respectively, for the orange 6 dye-binding method. EVALUATION OF POTATO PROTEIN BY AMINO ACID ANALYSIS AND DYE-BINDING BY Miklos Sandor Kaldy A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Food Science and Human Nutrition 1971 ACKNOWLEDGMENTS The author wishes to express his sincere apprecia- tion to Professor Pericles Markakis for his guidance throughout this study and for his aid in preparing this manuscript. He is also indebted to Professors Georg A. Borgstrom, Charles M. Stine, Norman R. Thompson and Walter M. Urbain for their advice and help in preparation of this manuscript. Appreciation is also extended to Miss Doris H. Bauer and Miss Ursula R. Koch for assistance in the amino acid determination and to Mr. Wojciech Malewski for as— sistance in colorimetric determination of protein. The author feels deeply grateful to the Canada Department of Agriculture, Research Branch, for the edu- cational leave and financial assistance granted to him during his studies at this university. ii TABLE OF CONTENTS LIST OF TABLES . . . . . . . . . . . . . LIST OF FIGURES O O O O I O O O O O O I 0 INTRODUCTION . . . . . . . . . . . . . . REVIEW OF THE LITERATURE . . . . . . . . Nitrogen in Potato . . . . . . . Proteins and Amino Acids . . . . Sulfur Containing Amino Acids . . Biological Value . . . . . . . . Protein Determination by Dye-bind ing MATERIALS AND METHODS . . . . . . . . . . Potatoes . . . . . . . .4. Freeze-drying . . . . . . . Total Nitrogen . . . .7. . Non-protein (Free) Nitrogen- Total Amino Acids . . . . . Sulfur Containing Amino Acid Tryptophan . . . . . . . . Free Amino Acids . . . . . Colorimetric Determination of in Potato . . . . . . . . . . Ninhydrin Method . . . . . Dye-binding Method . . . . oomogooo oooOoooon‘ooo O 0000”... D) H 'U ooofiooooj—looo +5 ff . geomooooaooo RESULTS AND DISCUSSION . . . . . . . . . Evaluation of the Potato Protein by Amino Acid Analysis . . . . . . . . . . . . . . . Sample Material . . . . . . . . . . . Moisture, Nitrogen and Sulfur Contents of Freeze-dried Potatoes . Total Amino Acids . . . Protein Scores . . . . Free Amino Acids . . . iii Sulfur Page vi 12 17 23 26 26 27 28 28 29 31 32 34 35 36 37 39 39 39 40 43 46 53 Estimation of "Total Protein" Colorimetric Methods . . Ninhydrin Method . . . Dye-binding Method . . S UM! O O O O O O O O O O Page in Potato by O C O I C O O O O O O 56 O O O O O O O O C O O 56 O O I O O O O O O I O 64 I O O O O O O O O O O 70 Evaluation of the Potato Protein by Amino Acid Analysis . . . . . . . . o o o o 0‘. o o o o o 70 Estimation of "Total Protein" in Potato by Colorimetric Methods . . REFERENCES . . . . . . . . iv 0 O I O C O O O O O O 72 O O O O O O O O O I O 74 LIST OF TABLES Table Page 1. Moisture, total nitrogen, "total protein," precipitable protein, total sulfur and sulfur present in S-containing amino acids . 41 2. Total amino acid composition of freeze—dried potatoes. (Grams of amino acids per 16 grams of total nitrogen.) . . . . . . . . . . 45 3. Essential amino acid composition of freeze- dried potatoes and whole egg . . . . . . . . 47 4. Protein scores of freeze-dried potatoes based on the essential amino acid pattern of whole egg . . . . . . . . . . . . . . . . 48 5. Protein scores of selected proteins based on the essential amino acid pattern of whole egg (FAQ/WHO, 1965) O O O O O O I O O O O O O 50 6. Comparison of the total amino acid composi- tion of potatoes reported by various authors. (Grams of amino acids per 16 grams of total nitrogen.) . . . . .,. .'. . . 51 7. Free amino acid composition of freeze-dried potatoes. (Grams of amino acids per 16 grams of non-protein nitrogen.) . . . . . . . 54 8. Comparison of the free amino acid composi- tion of potatoes reported by various authors. (Grams of amino acids per 16 grams of non-protein nitrogen.) . . . . . . . 57 9. "Total protein" content and ninhydrin color of freeze-dried potatoes . . . .,. . . . . . 62 10. Dry matter, "total protein" and absorbence at 480 nm of bound orange G dye in raw potatoes 0 O O . O I O O O O I O O O O I O O O 6 8 Figure 1. 2. LIST OF FIGURES Effect of concentration of ninhydrin on color development a o o o o o o o o o o o o ' o o 0 Effect of potato sample weight on the ninhydrin color development . . . . . . . . Effect of time of mixing by sonifier on the color development of ninhydrin with freeze-dried potatoes . . . . . . . . . . . Effect of time of boiling-on the color development of ninhydrin with freeze-dried pOtatOeS o o o o ' o o o o o o o a ' o o o o 0 Correlation between ninhydrin color and percentage of "total protein" (% N x 6.25) for freeze-dried potatoes. Correlation coefficient is + 0.9987. Standard error of estimate is 0.09% . . . . . . . . . . . Effect of concentration on the absorbance Of orange G dye o o o o o o o o o o 'o o o 0 Effect of time of shaking on the absorbance of orange G dye bound by raw potato . . . . Correlation between absorbance of bound orange G dye and the percentage of "total protein" for raw potatoes. Correlation coefficient is + 0.9827. Standard error of estimate is 0.07% . . . . . . . . . . . vi Page 60 60 61 61 63 66 66 69 INTRODUCT ION Proteins are fundamental in our diet and essential for good health. They provide the building blocks for the cells and the tools to direct body functions. However, unlike plants, animals can not synthesize all the basic elements needed for protein building. Consequently, ani— mals must resort to plants, either directly or indirectly, for these basic constituents. These basic constituents of proteins are the amino acids. Men and animals do not need proteins as such in their diet, but they do require some of the individual amino acids derived from them. There are 20 amino acids that are known to maintain the nitrogen equilibrium in the normal human adult. Eight of these are essential since they can not be synthesized by the body at a rate adequate to meet metabolic requirements. These eight must be sup- plied in the diet. Thus, the simple presence of proteins in a diet is not sufficient. They must supply a balanced amino acid mixture. The proportion of essential amino acids in a protein is decisive for its biological Value. When one or more amino acids are deficient (limiting) in a diet, the body does not make the protein without the deficient amino acid; rather the body makes less protein. As a result less protein is available for body building, repair and maintenance. Inadequate protein in the diet results in impairment of the health and may lead to death. Protein deficiency is a major nutritional and health problem in-the world today.- Kwashiorkor, a protein deficiency disease, is common in many developing countries. Since population growth in these countries may increase‘ more rapidly than food supply, the problem of protein deficiency is likely to become even more acute. This problem can be averted only by‘a combination cf population growth control, improved agricultural practices and eco- nomiC'development. The available animal protein is not enough to balance the world diet. Two-thirds of protein for world consumption comes from cereals (Borgstrom, 1967). Cereals, however, do not provide balanced proteins. In addition, the primary aim of past breeding practices was based on the misconception that quantity alone can feed the world. Yields, however, increase faster than protein, and man requires at least 12 percent of his calorie intake in the form of protein. Most of the world's early cereals con- tained 12 to 15 percent protein. In contrast, the present day's high yielding soft wheat varieties contain-only 10 percent or lower. Future agricultural research must not overlook the improvement of the nutritional quality of foods. Potato (Solanum tuberosum L.) has been in most cases by-passed as a source of protein. In its fresh state, potato has only an average of 2% "total protein." However, on a dry basis the "total protein" content of potatoes is not different from that of-wheat.r Also, an important fact overlooked by many is that one hectare of land under potato cultivation can supply the protein re- quirement for 9.5 people, while the protein of wheat from the same land can satisfy only 6.3 peOple (Borgstrom, 1969). Moreover, the biological value of potato for the human adult is 72 compared to 53 for wheat flour (FAO, 1957). Other determinations show biological value for potato as high as 78 (Chick and Slack, 1949) and 82 (Lindner gt_al., 1954). That the amount of protein present in the potato is sufficient for bodily needs was recorded as early as 1912 (Kelloqg, 1912). The Max-Planck Institute for Nutri— tion reported that when potatoes were consumed as the only source of protein, the daily protein requirement was an average of 0.55 g per kg body weight for man. Under the same conditions, when whole egg, milk, beef and tuna were consumed, the protein requirement per kg body weight were 0.51 g, 0.57 g, 0.57 g and 0.56 g respectively (Kofranyi, §E_al., 1965). Among these products egg supplied a better protein than potato. In comparison to the essential amino acids of the whole hen's egg, which has been adOpted for reference purposes by the FAO (1965), the sulfur containing amino acids are limiting in potato.- Potato is an easily digestible food, and a good source for Vitamin C. Its starch content (Kellogg, 1912), contrary to that in dry beans (Hellendoorn, 1969), under- goes no fermentation in the digestive tract. The high food value of potato has been proven in a large scale in Ireland.' From 1780 through the nineteenth century a large proportion of the population of Ireland became dependent for food almost entirely on the potato (Pyke, 1970). The consumption of potato, however, is responsible. for more than the sustainment of life. The dramatic in- crease in European population from 140 million-in 1750 to 400 million in 1900 is connected to the adoption of potato in man's daily diet (Langer, 1963). The dietary depend- ability of potato, therefore, was confirmed long ago. The degree of this dependability is not well de- fined. American research places the value of potato pro- tein lower than meat protein, whereas Eur0pean research claims it to be higher and closer to egg protein. The objectives of this research were to determine the distri- bution of total and free amino acids in potato with special emphasis on the limitation of the sulfur containing amino acids and to gain more knowledge about the value of the potato protein. In addition, a quick and simple method for determining the total protein of the potato was sought. REVIEW OF THE LITERATURE Nitrogen in Potato All living matter contains nitrogen. Nitrogen is one of the four major elements required in the cell build- ing process; the others are hydrogen, oxygen and carbon. Among these four major elements, the supply of nitrogen is the most restricted because man can not utilize nitrogen in all forms. Man can utilize ammonia to some extent but not nitrite, nitrate or N2. For the most part, nitrogen comes from dietary protein. Non—protein nitrogen such as free amino acids, nucleic acids, peptides and amides (glutamine and asparagine) can also be sources of nitrogen. In contrast to hydrogen, oxygen and carbon, nitro- gen is synthesized only into proteins and, to a small extent, into certain vitamins. The other three elements~ are widely distributed in all three classes of aliments, namely carbohydrates, fats and proteins. This restriction of nitrogen synthesis allows us to express protein in terms of nitrogen content (Hegsted, 1964). The total nitrogen content of a food protein varies from 15 to 19 percent, depending on its amino acid composition. For all practical purposes, with few exceptions, an average value of 16 percent nitrogen is arrived at. In most cases protein content is estimated by the wet combustion process which was originally devised by Kjeldahl (1883). This process is based upon the decomposition of the nitrogenous material, and the eventual release of reduced nitrogen in the form of ammonia from which, after titration, nitrogen is calculated. The nitrogen content is multiplied by 6.25 (100/16 = 6.25) for most foods to obtain the "total pro- tein" or "crude protein" content. Consequently, total protein nitrogen indicates both protein nitrogen and non- protein nitrogen. The ratio of these two components varies from product to product. In the potato the true protein nitrogen is_approx- imately 50 percent of the total nitrogen, but may vary, from 37 to 64 percent (Chick and Cutting, 1943; Neuberger and Sanger, 1942; Schuphan, 1960a). Protein nitrogen is by far the most important in delivering the necessary nitrogen for bodily needs. In some cases, however, as in potato (Kon, 1928), non-protein nitrogen plays an equally important role nutritionally (Mitchell, 1924a). To illus- trate: non-protein nitrogen of potato has no growth sup- porting capacity for rats. However, when 25 percent of non-protein nitrogen was replaced by potato protein (tuberin), growth became equal to that supported by. tuberin alone although somewhat lower than that sustained with intact potato (Chick and Slack, 1949). Apparently, non-protein nitrogen enhances the nutritive value of potato. Based on the observation of Rose and his co- workers (1948), the excess glutamic acid in the diet containing non-protein N in addition to protein stimulated growth.- Chick and Slack (1949) attributed the complemen- tary effect of non-protein nitrogen in potato to its glu- tamine content which they found to be twice as much as the protein nitrogen of the same potato. Glutamine and glu- tamic acid are very important in transamination reactions (Smith,‘1968). Proteins and Amino Acids The protein of potato is comprised of tuberin (a relatively soluble globulin) 76.4%, globulin II (less soluble than tuberin) 1.4%, albumin 4.0%, prolamin 1.8%, glutelin 5.5% and an unknown fraction 10.9% (Lindner,.eE a1., 1960). The variation in protein, as well as in dry matter content, among potatoes of different varieties, or among tubers of the same variety from different locations or sometimes from the same location, may be very large. Moreover, the nitrogenous compounds are not uniformly distributed over the tuber (Neuberger and Sanger, 1942; Mohler and Sulser, 1968). Solid content may vary from 15 t0 35 percent and "total protein" from 1.0 to 4.0 percent (Schuphan, gt_al., 1969). "Total protein" on a moisture free basis is inversely related to the solid content, but regardless of the solid content, the mg of nitrogen per g of fresh tissue is constant (Talley, et a1., 1961; Fithatrick, gt_al., 1964). "Total protein" content also varies with the conditions of culture and the variety of potato (Lampitt and Goldenberg, 1940; Jaschik and Lindner, 1955; Schuphan, 1960b). A heavy nitrogen fertilization greatly increases the protein content in potato (Mulder and Bakema, 1956; Schuphan and Postel, 1958; Nielsen, 1970). Westerlind (1970) reported increases from 50 to 120 percent in "total protein" with increasing nitrogen. supply to soil, but the essential amino acids decreased with this same increased supply. Mulder and Bakema (1956) found that phosphorus and potassium dressing greatly in- creased yields but drastically reduced "total protein." In addition, they discovered that the non-protein nitrogen content increased more than protein nitrogen when nitrogen dressing was increased, while the amino acid composition of potato protein was found to be independent of nitrogen, phosphorus and potassium nutrients supplied to the plants. In the case of valine, methionine and phenylalanine, the free amino acids contribute considerably more to the total amount of amino acids per unit of dry matter. The availability of amino acids determine the nutritive value of protein in a food. As early as 1912 an effort was made by Sjollema and Rinkes to analyze the amino acid content of potato protein. This early effort, however, is not very useful, probably due to the inadequacy of methods available at that time. Later, in 1945, Groot's investigation demonstrated low lysine and cysteine* content, but Slack's research in 1948 corrected Groot‘s low values and showed that methionine is present in potato in small amounts. Slack's investigation, however, like Groot's still limited the number of amino acids studied in potato to 11. Nevertheless, his results are very valuable as they are in good agreement with later efforts and permit comparison among protein nitrogen, non-protein nitrogen and "total protein" nitrogen (this third value was calculated from the first two). Later findings demone strated that protein nitrogen is richer in essential amino acids than the fraction of non-protein nitrogen (Chick and Slack, 1949). In 1949 Agren's determination indicated 18 amino acids in potato with low values for leucine, lysine, phenylalanine, threonine and cysteine. Lyman and Kuiken (1949) and Hirsch, gE_al. (1952) reported 10 and Wertz, gt_al. (1956) eight amino acids in potato, excluding cys- teine thereby reducing the value of the total sulfur con— taining amino acids. Eleven amino acids, including cys- teine were determined in potato by Schuphan (1960a). Thompson and Steward (1952) separated the total nitrogen *Some investigators report cystine and cysteine contents as cystine, others as cystine plus cysteine, others as half-cystine and still others as cysteine. Numerically the differences are insignificant among these values. In this thesis cystine plus cysteine will be reported as cysteine, which is a real building block of proteins. 10 into protein and non-protein fractions and determined their amino acid content but conducted no investigation on the total nitrogen. They found no correlation between the relative proportions of free and the protein bound amino acids. Their study included 15 amino acids and gave no values for histidine, tryptOphan and cysteine.' Mulder and Bakema (1956) also separated the total nitrogenous material into protein and non-protein fractions and determined in both fractions 18 amino acids with a combined value for leucine and isoleucine. Their values for lysine and cys- teine were relatively low. The low value for cysteine was not explained. Both methionine and cysteine were oxidized by ammonium molybdate to methionine sulfone and cysteic acid, respectively. Mulder and Bakema (1956) compared the free amino acids of two varieties of potato and the protein-bound amino acids of eight varieties of potato and found only small differences among the varieties. In contrast, the contents of protein nitrogen and non-protein nitrogen varied from 0.65 percent to 1.02 percent N of dry matter for protein nitrogen, and from 0.54 percent to 1.12 per- cent N of dry matter for non-protein nitrogen. Moreover, the ratio of protein nitrogen to non-protein nitrogen varied from 0.76 to 1.33. Eighteen amino acids were investigated by Hughes (1958) in potato protein and also in whole peeled potato 11 after being cooked by boiling. Composition of non-protein nitrogen was calculated from the values of protein nitrogen and the total nitrogen of whole cooked potato. Hughes' research on the protein fraction substantially agreed with Slack's (1948) in values for methionine and cysteine. Both indicated a total value of 4.4 g/l6 g N for the two amino acids combined. Bontscheff determined 14 amino acids with the application of paper chromatography.~ Talley and his co-workers (1964, 1970, 1970) stu- died the effects of storage, specific gravity, variety, location and year of growth on the free amino acid content of potatoes. They found an inverse relationship between total solids and free amino acids on a moisture-free basis, but there was little difference on the fresh-weight basis. Free amino acids changed randomly during storage. Nitrogen and free amino acid content varied according to location, but varietal differences were less significant. In respect to the year grown, aspartic and glutamic acids had the highest variability. The study by Sweeney and his co-workers (1969) supported Talley's finding that the free amino acids change randomly during storage, but states that these changes were greater in potatoes stored at 21°C than those stored at 13°C. Habib and Brown (1957) reported that the free amino acids of potatoes were greatly reduced after several weeks of storage at 24°C. This reduction of free amino acids is associated with protein synthesis. 12 When the tuber is in the rest period, protein is synthe— sized. At the end of the rest period, however, this process is reversed when hydrolysis takes place in the deeper tuber tissues. The released free amino acids then move toward the external (bud containing) tissues for protein synthesis in the buds because an active protein synthesis is required for the buds to sprout (Cotrufo and Levitt, 1958). These findings may explain the great variation in the ratio of protein and non-protein nitrogen in potatoes. Sulfur Containing Amino Acids One of the ironies of nature is that inorganic sulfur occurs abundantly in the earth, the oceans, and the atmosphere, while such important organic sulfur compounds as methionine and cysteine are in short supply (Rose, 35 31., 1954; Borgstrom, 1964). The scarcity of these sulfur containing amino acids is among the most crucial problems in human nutrition. Only plants can reduce sulfate, an oxidized form of sulfur, and synthesize methionine and cysteine (Thompson, et_al., 1970; Allaway and Thompson, 1966). Animals (and humans) can not perform the same reaction. Instead they depend on plants for the supply of methionine and cysteine. Both of these amino acids are important for protein synthesis, and of the two, methionine is essential for normal health (Rose, §E_al., 1948; Rose, 1949). Its prescursor, homoserine, can not be synthesized 13 from aspartic acid and must come from the food supply. On the other hand, unless obtained through dietary intake, cysteine can be formed from the ingested methionine and the non-essential serine. Consequently, dietary cysteine provides a methionine sparing effect. Cysteine is able to replace 80 to 89 percent of the methionine requirement (Rose and Wixom, 1955).. This sparing action is a result of the diversion of methionine from the catabolic pathway (transsulforation) to the pathways for protein synthesis and transmethylation (Finkelstein, 1970). The methionine supporting role of cysteine warrants the close study of both methionine and cysteine as the sources of organic sulfur compounds for the body. Methionine is the major source of methyl groups in the mammalian metabolism and, as such, is very important in transmethylation. Carnitine, a product of transmethy- lation, is an important participant in the mitochondrial fatty acid transport. Vitamin 312' epinephrine, ergosterol and lecithin are also end products of transmethylation- (White,g£_al., 1968). Organic S-compounds are building blocks in such compounds as S-adenosylmethionine, lipoic acid, coenzyme A, thiamine and biotin. They are essential for transmethylation, acetylation, CO2 transfer and the maintenance of the redox potentials in the cells (Finkel- stein, 1970). Furthermore, in bacteria, and also in the mitochondria of eucaryotic cells (e.g. liver parenchymal 14 cells), the synthesis of most, if not all, of proteins begins with N-formylmethionyl-tRNA, a formylated methionine tRNA (Clark and Marcker, 1968; Lehninger, 1970). Conse- quently, protein synthesis in these cells starts only when methionine is present. In recognizing the great need for methionine, efforts have been made to augment its supply in plant materials. Adequate sulfur nutrition in the soil in- creased the sulfur containing amino acids in absolute terms but not in relation to other amino acids. The proportion of the sulfur amino acids to the other amino acids remained largely unaffected; the protein, however, did increase due to the availability of an excess supply of sulfur to the soil (Thompson, gt_al,, 1960; Stewart and Porter, 1969). The ratio of protein nitrogen to non-protein nitrogen is greatly affected by the availability of sulfur. Stewart and Porter (1969) reported that protein nitrOgen accounted for less than 25 percent of the total nitrogen found in the sulfur deficient plants of wheat, corn and beans. In contrast, about 75 percent of the total nitrogen was protein when sulfur was adequate in these plants. They also demonstrated that the ratio of total nitrogen to total sulfur varied from about 4:1 to 55:1. Ratios of nitrogen to sulfur in the protein portion of wheat, corn and beans were, however, nearly constant with an average 15 nitrogen—sulfur ratio of 15:1. The nitrogen—sulfur ratios did not change with the variation in the amounts of nitro- gen and sulfur fertilizer added. Apparently, the amino acid composition of a given protein is constant and de- pendent only on genetical control (Stewart and Porter, 1969). Because of its consistency, the ratio of nitrogen to sulfur in food proteins may be used as a measure of the nutritional value of these proteins (Miller and Naismith, 1958; Miller and Donoso, 1963). It is proposed, however, that this method of evaluation be used only in mixed diets in order to avoid any anomalous results which may come from the interference of lysine, which can also be limiting in certain foods. The limiting nature of sulfur in animal products and potatoes, in contrast to cereal products, is apparent from the following nitrogen—sulfur ratios: milk 17.3:1, beef 15.1:1, potato 17.8:1, maize 11.6:1, barley 9.8:1 and oats 8.5:1 (Garrigus, 1970). These data indicate the limitation of the nitrogen—sulfur ratio when used for nutritive evaluation of foods. The application of the ratio, however, has definite advantages where the limiting amino acid is methionine in a food. Despite the great value attached to methionine, it is unfortunate that among the most common 18 amino acids, the sulfur containing amino acids are the most difficult to determine. Both methionine and cysteine are very 16 unstable during acid hydrolysis, especially in the presence of carbohydrates (Blackburn, 1968). Methionine is par— tially oxidized to sulfoxide, and cysteine is destroyed almost completely. Dustin, gt_al, (1953) and Schram, 22_ 31. (1953) investigated the effect of carbohydrates on 15 amino acids during acid hydrolysis, but they did not study methionine, cysteine and tryptophan, because they undergo drastic alteration or complete destruction under these conditions. There are good and relatively unlaborious chemical methods for the determination of tryptophan (Spies and Chambers, 1948; Block and Bolling, 1951; Spies, 1967), while at present, there are no rapid or simple methods for methionine and cysteine. In addition, the loss of amino acids in the presence of carbohydrates may vary from a small to a very large amount (Block, 1960). Based on earlier works of Toennies (1942) and Toennies and Homiller (1942), Schram and his co-workers (1954) partially over— came this difficulty by prior oxidation of cystine and cysteine residues with performic acid to cysteic acid which is stable to acid hydrolysis. This oxidation step was followed by the acid hydrolysis of protein and the quantitative determination of cysteic acid and other amino acids by ion-exChange chromatography. Schram's procedure, however, does not distinguish between cystine and cysteine residues present in the protein. Moreover, his is still a very time consuming process. 17 The development of a method for methionine deter- mination was also based on Toennies' (1942) work. Toennies had demonstrated that methionine is oxidizable by performic acid to methionine sulfone, which remains stable under acid hydrolysis. Applying this concept, Hirs (1956) oxi- dized both methionine and cysteine in ribonuclease to methionine sulfone and cysteic acid, respectively. The oxidation was followed by acid hydrolysis and determination of all amino acids, including methionine sulfone and cysteic acid, with ion-exchange chromatography. These methods and their modifications are used at present for the determination of methionine and cysteine, especially when interfering carbohydrates are present. When there is no interference from carbohydrates, the colorimetric test of McCarthy and Sullivan (1941) and its modifications are most commonly used. Biological Value Biological value is a measure of protein quality. The quality of a protein is in turn a measure of the effi- ciency with which it can provide for growth and maintenance of health. Thomas (1909) introduced the concept of bio- logical value, and expressed it as the percentage of retained and digestible nitrogen (N) from a test food. Mitchell (1924b) applied this concept for growing rats, and developed a method for determining the biological 18 value of protein with corrections for metabolic and endo- genous nitrogen. The basic calculation of Mitchell's biological value (BV) is: BV _ Retained food N - Absorbed food N x 100 Values for the two unknown functions of this equation are derived from the following considerations (Albanese, 1959): Absorbed food N = Food N — Fecal food N Fecal food N = Fecal N - "Metabolic" N of feces Retained food N = Absorbed food N - Excreted food N Excreted food N = Urinary N - "Endogenous" N. The values for "metabolic" N of feces and "endogenous" N of urine are obtained by measuring the nitrogen output of animals maintained on a nitrogen-free diet until a true nitrogen—minimum level has been achieved. The determination of biological value through feeding studies on animals or humans is time consuming and elaborate. Osborne and Mendel (1914) suggested that the nutritional value of proteins depends upon their content of those amino acids which cannot be synthesized in the~ body and are indispensable for normal metabolic functions. Their suggestion led to the conclusion that the nutritive value of a food protein can be predicted from the relative prOportions of the essential amino acids in it. 19 Subsequently, a method for the chemical evaluation of proteins based on amino acid content was developed (Mitchell and Block, 1946). This method is essentially a comparison of the essential amino acids present in a pro- tein with the prOportions existing among the amino acids of the whole egg which supports normal growth. A "chemical score" is then assigned to that amino acid which limits the nutritional value of the protein for maintenance and normal growth (Block and Mitchell, 1946). A chemical score is computed by deducting from 100 the percentage deficit in the limiting amino acid in relation to the same amino acid in egg protein. For example, the total sulfur containing amino acid content in whole egg is 346 mg/g N (FAO/WHO, 1965); in potato it is 138 mg/g N, and the deficit is (346 - 138)/346 x 100 = 60, which gives a chemical score of 100 - 60 = 40 for potato. This scoring, however, greatly underestimates the experimentally ob- tained biological values. The 1963 Joint Food and Agricultural Organization/ World Health Organization Expert Group (FAQ/WHO, 1965) recommended the adoption of the essential amino acid pattern of whole hen's egg for reference purposes when nutritive value is determined by chemical scoring. Ac- cording to the Expert Group "The content of each essential amino acid in a food protein is expressed as a percentage of the content of the same amino acid in the same quantity 20 of a protein selected as a standard. The amino acid show- ing the lowest percentage is called the limiting amino acid and this percentage is the chemical score." The FAQ/WHO Expert Group modified the computation of the chemical score to bring the score value in better agreement with the experimental biological value. It has been realized that whole egg provides more than double the estimated requirement of each essential amino acid than the minimal quantity that will maintain nitrogen balance in the adult body (Allison, 1958). Accordingly, a more realistic comparison between the quality of an unknown protein and the egg's protein is accomplished when the proportion of an individual essential amino acid of a food is compared to the corresponding amino acid in the whole egg (Sheffner, 2331., 1956; FAQ/WHO, 1965). The modified chemical score (protein score) is computed by summing up all the essential amino acids, in- cluding cysteine and tyrosine, to an amount not exceeding that provided by methionine and phenylalanine, respect- ively, and calculating the percentage proportion of the limiting amino acid to the sum of amino acids; these two steps are then followed by a comparison of this percentage with the corresponding one in the egg protein. According- ly, the total quantity of essential amino acids for man in potato is 2091 mg/g N. Of these, methionine and cysteine provide 138 mg/g N or 6.6%. The same values for whole egg 21 are 3215 mg/g N, 346 mg/g N and 10.8% respectively, and the modified chemical score for potato is 6.6%/10.8% = 61 when Orr and Watt's (1957) amino acid composition is ap- plied. In comparison, Bontscheff's (1965) amino acid composition provides a chemical score of 92% for potato. The biological value of potato, independently of methods applied for evaluation, shows great variations (Sheffner, 1967). Values obtained from experimental feed- ing studies varied from 60 to 82 (Lindner, et_al., 1954; FAO, 1957), and those calculated by chemical scoring varied from 61 to 92. The variations in biological value of potato are related to genetic features (Schuphan, 1960a). Tests on 15 potato varieties, cultivated in the same trial, indicated variations from 61 to 75 in biological value (Schuphan, 1958). Moreover, climatic conditions and fertilization also affected the biological value. The addition of nitrogen, phosphorus and potassium to the soil increased the biological value of potato, but an excess supply of nitrogen decreased it (Schuphan and Postel, 1958). Nevertheless, most of the investigators place the biological value of potato in the 78-80 range or higher. Kon and Klein (1928) maintained two persons (a male and a female) in good health with constant body weight for 167 days on a potato diet. This diet consisted of cooked potatoes, with or without oil, a few fruits, and 22 occasionally tea or black coffee with sugar. During the test the two subjects did not get tired of the uniform potato diet and showed no craving for change. The average daily protein intake per kg body weight was 0.560 g for the man and 0.384 g for the woman. A more recent study on human male subjects confirmed these earlier findings when Kofranyi (1965) demonstrated that .550 g potato protein per kg body weight was required to maintain nitrogen equilibrium compared to egg protein's .505 g, milk pro- tein's .568 g and beef protein's .565 g. Potato also has an excellent supplementary nature when consumed with egg. Only .380 g of 35% egg and 65% potato protein mixture were required per kg body weight to maintain the nitrogen equilibrium. On the contrary, the protein mixture of potato with milk, beef or tuna fish showed no marked im- provement above the values obtained from the same foods when consumed alone. Similarly, no supplementation oc- curred in mixtures of brown beans and potato, both being deficient in methionine (de Groot and van Stratum, 1963). The supplementary relations among proteins have been rec- ognized by Mitchell (1924c), and applied extensively for human nutrition by the Institute of Nutrition of Central America and Panama (INCAP) Guatemala (Bressani and Scrim- shaw, 1961; Bressani and Elias, 1968). 23 Protein Determination by Dye-binding Undoubtedly, the application of the quantitative reaction between proteins and anionic dyes at low pH to the measurement of protein content in various plant and animal products is a milestone in analytical chemistry. The significance of the event is emphasized by the move of the Association of Official Analytical Chemists to adopt as official the dye-binding method for determining protein in fluid milk (A.O.A.C., 1967). Based on the pioneer works of Loeb (1924), Chapman, §E_al. (1927) and Rawlins and Schmidt (1929; 1930) on the binding ability of dyes in buffered acid and alkaline solutions with protein groups of opposite ionic charge, Fraenket-Conrat and Cooper (1944) developed a micro-analy- tical method for the determination of the number of acid and basic groups of proteins. They learned that when proteins are treated with an excess amount of the anionic dye, orange G, at pH 2.2, they react stoichiometrically and form an insoluble complex. When this insoluble com- plex is separated by centrifugation or filtration, the concentration of the unbound dye can be measured with a photoelectric colorimeter.w The difference between added and unbound dye is the bound dye. The concept of the dye-binding capacity of protein was further develOped and applied for the determination of 24 "total protein" in cereal and milk products (Udy, 1954; 1956a; 1956b). Ashworth, gt_al. (1960) confirmed the reliability of the dye-binding method. Sherbon and Hemp- hill (1967) found that there was less variability among replicates analyzed by the dye-binding method than by the Kjeldahl method. The Kjeldahl method results, however, were not affected by the length of time lapsed between conducting duplicate determinations, while the results by the dye-binding method were affected. Gaunt and Gacula- (1964) reported that the spectrophotometric readings in the dye-binding procedure should be made within two hours from addition of the dye. Temperatures between 8° and 55°C had no significant effect on dye-binding (Ashworth and Seals, 1958). The best technique for predicting protein levels from the spectrophotometric measurements is to compare the absorbance rather than the amount of bound dye (mg ab- sorbed dye per g sample) with Kjeldahl analysis (Gaunt and Hankinson, 1969). In selecting a suitable dye, Dolby (1961) found amido black more sensitive for optical absorption, while Ashworth and Chaudry (1962) discovered that amido black showed less stoichiometry in reaction than orange G. In addition, amido black is unstable in solution (Sherbon, 1967), and deviates from Beer’s Law at concentrations above 6.2 mg/l (Steinholt, 1957). Orange G solution, on 25 the other hand, showed deviations from Beer's Law only at concentrations above 0.5 g/l (Ashworth, gt_al,, 1960). Further selection indicated that acid orange 12 is better for the dye-binding reaction because it has higher affinity for the protein and can be stabilized in solution by acetic acid (Sherbon, 1967). Acid orange 12, however, will bind free arginine if protein measurement is conducted in the presence of protein hydrolysate and considerable error may result. In contrast, orange G does not bind arginine (Seals, gt_al., 1969). The dye-binding method for the estimation of protein is rapid, and simple. It is used extensively for determining protein in fluid milk and cereal products.. It is also applicable to meat and egg products (Moss and Kielsmeier, 1967; Ashworth, 1971). The method is potenti- ally valuable in plant breeding for the selective work of high protein yielding varieties. No indications were ‘found in literature of the application of the dye-binding method for the determination of total protein in potato. MATERIALS AND METHODS Potatoes Random samples, 15 lbs. each, of the potato varie- ties Russet Burbank (RB), #58, #321-65, #322-6, #709 and #711-3 were obtained from the CrOp and Soil Science De- partment, Michigan State University in March of 1970. These varieties were grown during 1969 in Montcalm sandy loam soil fertilized by 128 lbs. N, 192 lbs. P205 and 192 lbs. K20 per acre. The potatoes were stored at 4°C at all times until sample preparation commenced. Samples from the six potato varieties grown in 1969 were freeze-dried and analyzed for total nitrogen ("total protein"), free nitrogen, total sulfur, total amino acids and free amino acids. The same samples were also used for the colorimetric determination of "total protein" in freeze-dried potato. Random samples, 8 lbs. each, of the potato varie- ties Abnaki, Bakeking, Cascade, Cobbler, Haig, Iopride, Jewel, Katahdin, Kennebec, B514l-6, Manona, Norchip, Pontiac, Russet Burbank, Sioux, Superior, #58, #321-65, #709 and #711-3, grown in 1970, were also obtained from the MSU Crop and Soil Science Department for the determi- nation of "total protein" by the dye-binding method. 26 27 These 21 varieties were grown on Montcalm sandy loam soil fertilized by 242 lbs. N, 112 lbs. P205 and 112 lbs. K20 per acre. Potatoes were stored at 4°C at all times until sample preparation for analyses commenced. Freeze-drying Fifteen pounds of unpeeled potato from each variety of RB, #58, #321-65, #322-6, #709 and #711-3, were sliced by a potato slicer to 1 mm thickness, and then quick-frozen in stainless steel trays containing alternate layers of dry ice to prevent browning. Dry ice was removed (subli- mated by placing the trays with the frozen potatoes into a freezer set at 520°C. The slices were freeze-dried in a Virtis REPP Model FFD42 WS freeze drier at a plate temperature of 38 to 80°C with a vacuum of less than 5 u. Before freeze- drying commenced, product temperatures were reduced to -25°C. The freeze-dried potato was broken up and sieved through an 8 mesh sieve to remove peel and scar tissue, and then stored at -18°C in screw tOp jars for analysis. Prior to the analysis, a portion of the frozen samples was sieved through an 80 mesh sieve, facilitated by pre-grinding in a mortar where particle size made this necessary, and stored at 4°C. New samples were prepared at weekly intervals to avoid possible decomposition. 28 Total Nitrogen The conventional "total protein" content (N x 6.25 on a dry matter basis) of the freeze-dried potato was determined by the A.O.A.C. (1970) micro-Kjeldahl method with sample weights of 0.15 g. For dry matter determina- tion samples were dried to constant weight in a vacuum oven at 70°C. The "total protein" content of raw potatoes was determined by the A.O.A.C. (1970) Kjeldahl method for plants.. Sample weights and sodium hydroxide solutions were 10 g and 75 ml, respectively. For the analysis four randomly selected, peeled potato tubers were cut into 0.5 x 0.5 cm dices. From the same diced potato 15 g samples were dried to constant weight in a vacuum oven at 70°C, and the dry matter contents were calculated. Non- rotein (Free) Nitrogen and Tota Sulfur One gram freeze-dried potato was mixed with 10 ml of 10 percent trichloroacetic acid (TCA), left to stand for one hour, and then centrifuged. The supernatant was decanted and saved, and the precipitate was washed twice with 5 ml 5% TCA and centrifuged. The collected super- natants were concentrated in a rotary evaporator under vacuum to about 5 m1, then diluted to 10 ml with 5% TCA. One and a half milliliters of this solution were used to determine the non-protein nitrogen content by the 29 microéKjeldahl method. Protein—nitrogen content, on the other hand, was obtained by subtracting free nitrogen from the total nitrogen. Total sulfur was determined by the A.O.A.C. (1970) magnesium nitrate method recommended for plants. Total Amino Acids The analyses for total amino acids in freeze-dried potatoes were performed on 22 and 72 hours hydrolysates of the protein in the freeze-dried samples. For these anal- yses a Beckman Model 120C Amino Acid Analyzer was employed in which the amino acids were separated by column chroma- tography, and quantitatively determined by automatically recording the intensity of the color produced by their reaction with ninhydrin (Moore and Stein, 1948; 1951; 1954; Moore, gt_al., 1958; Spackman, g£_al., 1958). Samples were prepared in accordance with the Beckman manual for amino acid analysis (Toepfer, 1965) as applied by Rhee (1969). About 25 mg portions of freeze-dried potato were weighed directly into 10 m1 ampules. Five milliliters of glass-distilled 6 N HCl were then added to the ampules. The contents of the ampules were frozen in a dry ice- alcohol bath, evacuated with a high-vacuum pump, and al- lowed to melt slowly under vacuum to remove dissolved gases. After the samples had been fully evacuated, the ampules were sealed with a pin-point air-prOpane flame. 30 The evacuated and sealed ampules were then placed in an oil bath set in a 110:1 C°oven. After hydrolysis of 22 and 72 hours, the ampules were removed from the oven, and cooled first to room temperature and then to 4°C in a refrigerator. After cooling, the tOp of the ampules was removed by making a deep scratch with a triangular file on the neck of the ampule and by breaking the neck with gentle force. One milliliter of 2.5 uM norleucine solution was added to each ampule as an internal standard to measure transfer losses. The contents of each ampule were trans- ferred to a pear-shaped evaporating flask, and evaporated to near-dryness under vacuum on a rotary evaporator im- mersed in a 55°C water bath. After evaporation a small amount of deionized water was added to the residue which was then resuspended for evaporation. This process was repeated twice more, or until all remaining hydrochloric acid residue was removed. The dry film of hydrolysate was dissolved in about 3 ml buffer (0.067 M citrate-hydrochloric acid buffer, pH 2.2), and then transferred quantitatively to a 5 ml volu- metric flask. The pear-shaped evaporating flask was washed twice with buffer, and the final volume was made up to 5 ml. In order to separate the carbohydrates, the 5 ml hydrolysate was centrifuged for 10 minutes on.a clinical centrifuge, and the clear supernatant was transferred into 31 a small screw top vial and stored at 4°C. An aliquot of 0.4 ml from the stored supernatant was then transferred to the analyzer for analysis. The amino acid analyzer was Operated at 57°C, and the running time was four hours (55 minutes for the short and 185 minutes for the long column). The resulting chromatograms were compared to those obtained from the analysis of a standard amino acid cali- bration mixture. The ratios of areas under the curve for the samples and the standards were compared and converted to give the amino acid composition of the sample as gram amino acid per 100 grams of protein (Toepfer, 1965). Sulfur Containing Amino Acids To protect cysteine and methionine against loss during acid hydrolysis when carbohydrates are present (Schram, gt_al., 1953), cysteine was oxidized with per- formic acid to cysteic acid, and methionine to methionine sulfone as described by Schram, gt_al. (1954) and Lewis (1966). The performic acid reagent was prepared by mixing one volume of 30% (w/w) hydrogen peroxide with nine volumes of 88% (w/w) formic acid. The solution was allowed to stand for one hour at room temperature for maximum forma- tion of performic acid. Six hundred milligrams freeze- dried potato samples were then weighed into 200 ml jars with narrow bottoms, and the jars with samples were cooled to 0°C in an ice bath. Next, 10 m1 of performic acid, 32 cooled previously to 0°C, were added to the samples in the jars so that all material came in contact with the solu- tion. Oxidation was allowed to continue for 16 hours at 0°C, a temperature maintained with an ice bath kept inside the refrigerator. At the end of the oxidation period performic acid was removed by freeze-drying after 20 ml of ice-cold water were added to the samples. The freeze-dried residues from the oxidation were hydrolysed, as described previously, with 5 m1 glass- distilled 6 N HCl for 22 hours. The hydrolysates were then freed from hydrochloric acid, diluted to 3 m1 and centrifuged; 0.3 m1 aliquots of the supernatant were analyzed on the Beckman Model 120C Amino Acid Analyzer. The resulting chromatograms were compared to that of standard methionine sulfone; in addition the ratios of areas under the curve were compared, converted to amino acid values and expressed as gram amino acid per 100 grams protein. To calculate cysteine the values of the chroma- tograms' curve were compared to the height-width (HW) constant, obtained by multiplying the "average standard HW constant" with 0.97, a factor recommended in the Beckman manual (Toepfer, 1965). Tryptophan Trypt0phan is subject to destruction during the acid hydrolysis of proteins. Consequently, this amino acid must be determined separately. Tryptophan was 33 determined spectrOphotometrically after hydrolysis with pronase as described in Procedure W by Spies (1967): (l) (2) (3) (4) A 30 mg freeze-dried potato sample was weighed directly into a 2 ml glass vial with screw cap. To each glass vial 0.1 ml (100 pl) of pronase hydrolytic solution and a drop of toluene, as a preservative agent, were added. Pronase solution was prepared daily by the addition of 100 mg pro- nase to 10 m1 of 0.1 M phosphate buffer, pH 7.5. The suspension was shaken gently for 15 minutes, and then clarified by centrifugation. The vials were closed, and then incubated for 24 hours at 40°C. At the end of incubation 0.9 ml of 0.1 M phosphate buffer, pH 7.5, were added to each vial. After uncapping the vials, and removing pencil marks from their exteriors, they were placed into 50 m1 Erlenmeyer flasks containing 9.0 m1 of 21.2 N sulfuric acid and 30 mg p- dimethylaminobenzaldehyde (DAB). Next the vials were tipped over and the contents quickly mixed by swirling. Samples were cooled to room temperature, and placed in the dark at 25°C for 6 hours. To each flask 0.1 m1 of 0.045% sodium nitrite was then added. After gentle shaking, 30 minutes were allowed for the development of color, followed by the measurement of the absorbance of the solution 34 at 590 nm. The blank solution contained everything but protein and pronase. Duplicate samples of the pronase hydrolytic solution without sample mater- ials were run similarly, and the tryptophan content of the pronase was subtracted from the total tryptophan contents. A standard curve for zero to 120 pg of tryptOphan was prepared in accordance with Procedure E by Spies and Chambers (1948): 2.4 mg tryptophan were dissolved in 200 ml 19 N sulfuric acid containing 600 mg DAB. From this solu- tion 0, 1, 2, 4, 6, 8, and 10 milliliters were supple- mented to 10 ml with solutions of 19 N sulfuric acid containing 600 mg DAB/200 m1, and placed in 50 m1 Erlenmeyer flasks.r Flasks with the reaction mixtures were placed in the dark at 25°C for 6 hours. Then 0.1 m1 0.040% sodium nitrite was added to each flask.' After the 30 minutes allowed for color develOpment, absorbances were read at 590 nm. A straight line relationship was obtained between absorbance and amount of tryptophan. Free Amino Acids The free amino acid content of the six freeze-dried potato varieties was determined with a Technicon Amino Acid Analyzer. Samples were prepared as described by Toepfer (1965) for tissue extracts. 35 A 3.5 g potato sample was triturated with 35 ml 1% picric acid, and the mixture was promptly centrifuged to remove the precipitate. The supernatant liquid was then passed through a 2 cm high Dowex 2—x1o resin column in a 2 x 20 chromatograph tube. The walls of tube and the resin bed were washed with five 3-m1 samples of 0.02 N HCl. The effluent and washings were concentrated under vacuum on a rotary evaporator at 55°C to about 3 ml. The concentrate was quantitatively transferred to a small glass tube and adjusted to pH 7.2 with l N NaOH. To this solution 0.2 m1 of freshly prepared 0.5 M sodium sulfite was added, and the sample was allowed to stand, Open to the air, for four hours. The pH of the solution was adjusted to 2.2 with 1 N HCl, and diluted to 10 ml final volume. Thirty to fifty microliters of each sample were analyzed on the Technicon Amino Acid Analyzer. Free amino acids were calculated in a similar manner as total amino acids. Colorimetric Determination of "Total Protein“ in Potato Two colorimetric methods were developed for the estimation of "total protein" in potato. One is based on the development of color by the ninhydrin as it reacts with the free amino groups of amino acids, peptides and proteins in the freeze-dried potato. The second 36 colorimetric method is based on the absorbance of orange G dye by proteins in raw potatoes. Ninhydrin Method Six varieties of freeze-dried potato were analyzed for "total protein" by the following method: (1) (2) (3) (4) (5) (6) Exactly 500 mg freeze-dried potato, calculated as dry matter, were weighed into a 50 ml polyethylene centrifuge tube. To the sample 25 ml of 0.015% ninhydrin solution were added. Ninhydrin solution was prepared weekly by the addition of 0.015 g reagent grade ninhydrin to 100 ml 0.1 M citrate buffer, pH 4.4, and stored in the refrigerator. Sample and ninhydrin solution were then mixed by a sonifier (Model LS 75, Branson Instruments, Inc., Stanford, Connecticut) for 1.5 minutes. (Settings: direct current was 4 ampers and the power setting was 2.) The centrifuge tubes were placed into boiling water, and boiled for 20 minutes. Tubes were cooled in a water bath, placed into the refrigerator and allowed to stand for 10 minutes to develop full color. Contents in tubes were then centrifuged for 15 minutes using a SS Sorvall centrifuge. 37 (7) Finally, the absorbance of the supernatant was read at 570 nm (Clark, 1964) against the blank. The blank contained no sample material. The absorbances were plotted against the Kjeldahl protein values, and a regression equa- tion, correlation coefficient and standard error of estimate were calculated (Steel and Torrie, 1960). Dye-binding Method Twenty-one varieties of raw potato were analyzed for "total protein" by the dye-binding method, using the dye orange G: (1) (2) (3) Three randomly selected potato tubers were peeled and cut into 0.5 x 0.5 cm dices. Two grams of this sample were weighed and trans- ferred into a 15 ml tapered glass tissue grinder (TRI-R Instruments, Inc., Rockville Centre, N.Y.) and ground by hand until all cells were broken. The grinding required about one minute of work. The ground sample was then transferred in portions to a 50 ml centrifuge tube with a total of 25 ml dye solution. The dye solution was prepared by dissolving 0.02 9 orange G, with 94% dye content, in 1,000 m1 of the following buffer: 3.4 g KH P04, 6.8 ml 2 H3PO4 (1 part 85% H P04 + 1 part H20), 60 m1 HOAc, 3 (4) (5) (6) 38 1 ml prOpionic acid and 2 g oxalic acid in about 800 m1 H O, adjusted to pH 1.8 and then diluted to 2 1000 ml with H O (A.O.A.C., 1967). 2 Sample and dye solution were then vigorously shaken end to end for 10 minutes in a mechanical shaker which was set at 250 strokes per minute. The contents of the tube were centrifuged for 10 minutes using a SS Sorvall centrifuge. The absorbance of the supernatant was read at 480 nm against water. The difference in absorbance between the original dye solution and the super- natant of the reaction mixture was taken as a measure of the bound dye. The values for bound protein were plotted against the Kjeldahl protein, and a regression equation, correlation coefficient and standard error of estimate were calculated (Steel and Torrie, 1960). The absorption spectrum of orange G was meas- ured with the 505 Bausch and Lomb spectrOphoto- meter, and 480 nm was found to be the absorption maximum for the dye. RESULTS AND DISCUSSION Evaluation of the Potato Protein by Amino Acid AnalysIs Sample Material The difficulty that exists in maintaining fresh potatoes in unchanged conditions for a continued study necessitated the storage of potato samples in a more stable form, a form which was obtained through the freeze- drying process. During freeze-drying water was removed from the potato in a frozen state. The effect of this type of dehydration is minimal to the product because the reaction rates are low at reduced temperatures. Chick and Cutting (1943) reported that when mashed potatoes were dried at 40°C, the nutritive value of the nitrogen of the dried material was identical with that of freshly steamed and skinned potatoes. Nevertheless, browning, which is characteristic of cut potatoes, may have occurred at 40°C of drying temperature. No browning occurred in the freeze-dried samples prepared for the present study. 39 40 Moisture, Nitro en and Sulfur Contents 0f Freeze-drie Potatoes The percentages of moisture, total nitrogen, "total protein" (% N x 6.25), protein nitrogen.and sulfur in the freeze-dried potato are given in Table 1.. The moisture contents of the samples varied from 2.80% to 5.58%. In order to prevent possible changes at these moisture levels, the dried potato samples were stored at -18°C. The total nitrogen contents of the dried potato samples varied from 1.29% to 1.96% on percent dry weight. These results are similar to those reported by other authors (Neuberger and Sanger, 1942; Chick and Slack, 1949; Hughes, 1958). "Total protein" contents (% N x 6.25) of the samples varied from 8.1% to 12.3% on percent dry weight. The protein nitrogen, expressed as percentage of total nitrogen, was obtained from both trichloroacetic acid (TCA) and picric acid (PA) precipitates. Free amino acid determination required the precipitation of protein by 1% picric acid (Toepfer, 1965); precipitation of protein by 10% TCA was also used to obtain values for comparison. The results of these two determinations are not in good agreement. Apparently, 1% picric acid precipitates more nitrogenous substances (59.5% to 85.3%) than 10% TCA (29.7% to 54.1%). Moreover, the precipitates of the two 41 moans osflfid I .m.¢ owns oauowm I 4m owom owuoomouoHcoHHB «OB xsmnusm pmmmsm s no.o mo.o ao.o ao.o oo.o mo.o ao.o A.us use no we .¢.¢ mswcflmus00|m cw usmmmum m mH.o ma.o sa.c HH.o NH.o ma.o 4H.o A.u3 use no my pmeasnmump m Hmuoe oo.He om.am om.em o~.~a om.mm om.mm oa.ea am An pmnmunaaomua oo.ss om.ms oa.sm oa.mm oo.mq os.ms oa.ss sue an cosmonanomud z Hmuou mo mmmusmouom om.oa om.aa oa.m om.m oa.m om.ma om.oa A.u3 who no we Am~.o x 2 my =camuoum Hence: mm.a om.H ms.a mm.a mm.a mm.a on.H 1.»; use no we 2 Hence sa.m mm.m .om.m mm.m o~.m om.m om.~ Awe musnmaoz .m>¢ muaaae mane mummme mmuamme mme «mm mowuowum> .moaom ocwem msflswmucoclm cw usommnm “Sufism use Homasm Hmuou .cflmuoum manmuflmwomum.xcwouonm Hmuou=.smmouufls Hmuou .musumfloz .H magma 42 acids are not proportional to each other when the contents of varieties are compared. Neuberger and Sanger (1942) investigated the non- protein fraction of potato, and reported that on the average 50% of the non-protein nitrogen was present as amides (glutamine and asparagine), 35% as nitrogenous bases (arginine, purines, choline) and 15% as a-amino acids. Thompson and Steward (1952) indicated that gluta- mine and asparagine amounted to over 75% of the non-protein nitrogen fraction in potatoes. The nitrogen values of the TCA precipitates determined in this study are in good agreement with values reported by other authors (Neuberger and Sanger, 1942; Chick and Cutting, 1943). Determination of total sulfur was conducted with the expectation that potato varieties can be evaluated for the critical sulfur containing amino acids on the basis of total sulfur content. The six potato varieties analyzed contained from .11 to .18% total sulfur on the dry weight. Unfortunately, the total sulfur values are not in good agreement with the sulfur values calculated from the methionine and cysteine content of the sample. Numerous methods have been suggested over the years for the deter- mination of sulfur, which indicates that this seemingly simple analysis still causes great difficulties. 43 Total Amino Acids The total amino acids of the six potato varieties were calculated in micromoles and expressed as gram amino acid per 16 grams of total nitrogen (Table 2). With the exception of methionine, cysteine and tryptOphan, all amino acids were determined from 22 and 72 hours hydroly- sates. During acid hydrolysis some losses of histidine, threonine and serine occurred. The values for these amino acids were corrected for the loss (extrapolated to zero hydrolysis time) according to the equation given by Hirs, §E_al. (1954): t t1 ° (log A ) - _ 1 1 t2 t log A0 = (log A2) 1 where A1, A2, A0 are the quantities of amino acids after t1, t2, and to hours of hydrolysis, respectively. The times of t1 and t2 correspond to the 22 and 72 hours of hydrolysis, respectively. All other amino acid values were calculated as the average of the 22 and 72 hours of hydrolysis. Tryptophan was destroyed during acid hydroly- sis, and was determined separately by the method of Spies (1967). In order to protect methionine and cysteine from destruction during acid hydrolysis, these amino acids were oxidized before hydrolysis to methionine sulfone and cysteic acid, respectively, by performic acid (CHZO3): 44 NH NH 2 CH203 fi ' 2 CHB-S-CHZ-CHZ-f-COOH + CH3-fi-CH2-CH2-C-COOH H O H methionine methionine sulfone OH fH 0-=S==O CH In I 2 CH203 I 2 H--(|I--NH2 #% H- -NH2 COOH COOH cysteine cysteic acid when the oxidation was completed, the samples were acid hydrolysed for amino acid analysis. The six potato varieties showed similar patterns of amino acid composition. The deviations of the amino acid values between varieties varied from the average by i4.l% for arginine and i29.5% for serine at the extremes. The mean variation was i17.0%. Methionine and cysteine showed variations of 312.5% and :15.0%, respectively. Seem- ingly, these variations are not large, but a 10 to 15% change in the sulfur containing amino acids may alter the protein quality of a product significantly. The total amino acid nitrogen (NH3 included) was calculated for each of the six varieties, and then compared ' f freeze-dried potatoes. (Grams 101'). 0 d composit ds per 16 grams of total nitrogen.) 1110 am. Total am Table 2. ino ac1 of am Varieties #321-65 #322-6 #709 #711-3 Ave. #58 RB* Amino acids 'U «4 O ('6 (I) (D {360:1 "-1 Q-I-l-r-l (D'U-r-l-IJQ' SWISS-IO w-l-IJ-v-ICUCD (010010434 h-Hémfi as: 426-! I—Ifi'o-ION £0va N mmcnm \ONV‘M N 18.6 I‘V'O‘OQ' LONQ‘ONM N mmm 5 2 4 28.9 3.6 Serine Q'Ln nun r-l Glutamic acid Proline Glycine 45 l‘kONOl‘lnln mvamvH OKONkOV‘MQ‘ \Dt-IVLD fl'r-l l‘l‘MI-IFFLO C LnI—IV'KDMV'l—I O‘Q‘fl'mOAU-IKD mu—vaommr-I Lnfl'meNl‘ LfiH'fl'l‘fl'LfiI—I V‘QKOO'Q'OWM MNNf-ILOHMLDMMt-l Alanine Cystine + Cysteine CDLDO\ml\O\l\ Lnr-IMV'MMI-I o c: -a (DO) ctr: c a old -A«4 or4: cowards. OOSG-Hv—IO szvio-acnanu mucr4L)o ssh r4+)o:3Lao:» monomcu >>2I4+JE4QIB Total amino acid N 82.1 80.5 77.3 83.0 79.9 79.2 77.4 as % of Kjeldahl N *Russet Burbank 46 to the corresponding Kjeldahl nitrogen. The recovery of the Kjeldahl nitrogen varied from 77.3% to 83.0%, with an average of 80.2%. The unaccounted for 19.8% nitrogen could be attributed to amides, purines, pyrimides, choline, some vitamins, etc. Protein Scores The essential amino acids of the six potato varie- ties were compiled and expressed as milligrams of amino acid per gram of total nitrogen (Table 3). These values were compared to the essential amino acid composition (pattern) of whole egg, and the protein scores were cal— culated (Table 4) as described by the Joint FAQ/WHO Expert Group (1965). Protein scores were calculated with the inclusion of the non-essential tyrosine and cystine + cysteine amino acids which have sparing actions on phenylalanine and methionine, respectively. This sparing action occurs be- cause the body forms tyrosine from phenylalanine and cysteine from methionine, when tyrosine and cysteine are not present in sufficient amounts. Naturally, tyrosine and cysteine can be included only in amounts not exceeding those of phenylalanine methionine because the body can not convert tyrosine to phenylalanine and cysteine to methionine. The protein scores verified earlier reports (Slack, 1948) that methionine is the most limiting amino 47 om3\omm Sony ma sumo moo oHocz .Ammmav .mam>fluoommou .ocwcoOnuofi can ocflsmamamconm an ooow>oum poop msflooooxo nos mussosm GO omosaosfl one ocOoumao + msflummo com ocwmouma «s xsmousm ummmsm « OHNO OHON ONON OOON OOON ONON OOHN OHNN .a.« HOHpemmmm Hence OOO OOO OOO OOO OOO OOO OOO OOO meHHO> OOH NO OO OOH OOH OOH HO OOH ameaosasue OHO OON OHN OHN OON OOO OHN ONN meHcomuae OOH OO OO mm mm mm OO ON mchumOo + meHuOOu OOH NO OOH OOH OO OO OHH OOH mchoHeumz OOO OOH OOH OOH OOH OOH NOH OOH .a.¢ .peooum Hmuoe NON NON NHN HON OON OON NHN HON OOmchonOe OOO OON OON OON OHO ONO OON OON meHeOHOHOemam ONO OHO HOO ONO OOO OOO OOO OOO .<.a =6Humsoum= Hmuoe OOO OOO HOO OOO OOO OHO OOO HOO OOHOOH OOO OOO OOO HOO ONO OOO OHO OOO meHosmH OHO OON OON OON OON OOO ONN OON ocHoOmHomH mom mommm>< OIHHOO OONO OINNOO OOIHNOO OOO OOO A.O.NO OOHom oeHea mHonz moquOHm> comouuflc Hmuou mo Emma Mom moflom osOEo mo memumflaaflz .mmm oaoc3 can mooumaom Umwnolonomum mo soOpOmomEoo oflom ocOEm HMOusommm .m magma 48 xcmnnsm ummmsms OOHA OOHA OOHA OOHA OOHA ooaA OOHA mumnuo HH¢ OOHA OOHA OOHA OOH mm OOHA OOHA OCHHM> ooa mm om OOHA OOHA mm OOHA OCHGOOHSB Hm mm Hm mm OOHA Om mm OGHOSOQ mm mm mm Hm mm Hm mm OGOOSOHOmH mm mm mp mm om mm mm .¢.< .ucOO Hflmadm .m>¢ mlHH5¢ mon¢ mINNm* mmlammw mm¢ smm A.d.¢v MUHO¢ OGHE¢ wwflfimflHMKV Hafiucwmmo oz» .mmo oaoc3 mo snouumm oOom ocOEm co comma mooumuom coauolouooum mo mmuoom samuoum .v manna 49 acid in potatoes. Methionine is followed by leucine, isoleucine, valine and threonine in limiting action. The remaining amino acids have protein scores greater than 100. The protein scores for the potato varieties Russet Burbank, #58, #321-65, #322-6, #709 and #711-3 are 73, 78, 60, 62, 73 and 68, respectively. The average protein score for the six varieties is 69. Variety #58 has the highest and #321-65 the lowest protein quality among the tested varieties. The protein quality of Russet Burbank, a well accepted commercial variety, is better than average. While the nutritional value of protein is basically deter- mined by the amino acid composition, protein scores predict only the utilization of protein after absorption. If one or more of the essential amino acids are unabsorbed, the biological test would probably give lower values for the same food. However, the protein scores are still reliable measures for the quality of proteins, and provide at the same time a means of comparison with other food proteins. The protein scores calculated by the FAQ/WHO Group (1965) for a variety of protein sources using the essential amino acid pattern of egg are listed in Table 5. A comparison of the average values of the total amino acids in the six potato varieties investigated in the present study with those reported by other authors for whole potatoes is shown in Table 6. Other investigators have published on the amino acid composition of fractionated potato proteins. 50 Table 5. Protein scores of selected proteins based on the essential amino acid pattern of whole egg (FAQ/WHO, 1965). Food Protein Score Milk (cow's) 60 E99 100 Casein 60 Beef muscle 80 Pork tenderloin 80 Fish 75 Oats 70 Rye 90 Rice 75 Corn meal 45 White flour 50 Soy flour 70 Potato 70 Navy been 42 Potato in this study 69 51 m.a N.O w. 0.0 H. m.H m. cmnmoumhua 0.0 0.0 0.0 O.O 0.0 N.O O.O mcHemHmHOcmam b.m m.N m.H I I I I mswmouaa 0.0 0.0 . >.O m.v 0.0 . msaosoq N.O m.m m NH m.m m.O h.m m HH ocflosoaomH 0.0 m.H w.H O.H m.a m.H O.H osOGOOcpoz >.m m.O o.m m.m m.m m.O m.v msOHm> m.H m. m.H m. I I >.H oswmumhu + ocflummu H.m m.m I I I I I oswsmad o.m m.m I I I I I mcwomau m.m H.m I I I I I mcflaoum m.mH H.mH o.m I I I I oOom owemusaw 0.0 h.m 0.0 I I I I ocflumm m.m m.m o.~ H.O m.m m.~ h.m osacoouca m.vm m.vm m.oa I I I I owes oauummmd 0.0 0.0 0.0 N.O 0.0 0.0 0.0 mchHmus m.~ 0.0 O.H o.~ m.H O.H h.H osOoOUmOm N.O v.m m.m N.O m.m N.m o.m ocOmmH Oesum AOOOHO AOOOHO AOOOOHO AMMOHP AOOOHO AOOOHO OOHoa ocHea macs Homom mmocomusom smcmscom .am no coxasx xUMHm Iuouoscom comuflm can cmfihq H.2omouuwc Hmuou mo macho ma mom moflom osOEm mo mfimnwv .muosusm msoHHm> an omuuomou mooumuom mo coOuOmomEoo cOom osOEm Hmuov may no GOmOHmmfiou .O manna 52 The amino acids in this study, with the exception of cysteine, are in good agreement with the recent invest- igation of Schwerdtfeger (1969). Cysteine was not oxidized to cysteic acid in the latter work, and, consequently, the greater part of cysteine was lost during hydrolysis. Interestingly, the seemingly high aspartic and glutamic acids reported in this study are almost identical with those reported by Schwerdtfeger (1969). For a better comparison of the various amino acid compositions listed in Table 6, protein scores were cal- culated in this study only in those instances where cystine + cysteine was reported. The protein scores based on the essential amino acid patterns given by Slack (1948), Schuphan (1960a), Bontscheff (1965) and Schwerdtfeger (1969) were calculated to be 87, 59, 92 and 54 for sulfur containing amino acids, respectively, and 74, 100, 71 and 100 for tryptophan, respectively. The wide divergence of results probably is due to differences in methodology rather than actual dif- ferences in the samples analyzed. Bontscheff used paper chromatography which can not be considered as accurate quantitatively as the ion exchange chromatographic method. The amino acid composition of the "total protein" of potato was calculated by Slack (1948) by combining the values obtained for the amino acid content of the non- protein fraction with those found for tuberin. Slack 53 determined methionine and cysteine colorimetrically without the oxidation of these acids to methionine sulfone and cysteic acid, respectively. Similarly, Schuphan (1960a) and Schwerdtfeger (1969) reported no oxidation procedure for methionine and cysteine, and the protein scores based on their investigations were 59 and 54, respectively, when calculated by the FAO/WHO (1965) method. Free Amino Acids Since previous investigators (Kon, 1928; Chick and Cutting, 1943) had observed that the non-protein nitro- genous constituents of potatoes are well utilized by the body, the determination of free amino acids in potatoes was warranted in this study. The free amino acid compo- sition of the six potato varieties Russet Burbank, #58, #321-65, #322-6, #709 and #711-3 are given in Table 7. In contrast to the total amino acids the variation of free amino acids among potato varieties are very pronounced. Also, there is no proportionality between the free amino acids and the total amino acids of the potato. Similar variations of free amino acids were observed by Mulder and Bakema (1956). They reported that "the composition of the soluble non-protein fraction is much less constant than that of the protein fraction. It is affected by the mineral nutrition, variety, and presumably by other fac- tors like climatic conditions, age, etc." In contrast, mm commmnmxo ma Edm “Hose pmuomume uoz«« .ocanom .xmoo moo mm condemns msfluom can ocwcooucas xsmnnsm ummmSM+ 54 0'40”)me C O O O O O O C O O V‘NONFFH NkO NMI-Imm Q'O‘r-IONOl‘t-‘lmq'm O \DOQNV' o o o o o o o o o o o o o 0 Na) I—lvl-{MV' mmrxcsoomuo ommmmmxovzrhm N0 FINI-INQ‘ I-lInr-I Homooxo it '3 N O O N h.HN M 0‘ r-I N.HH m.N m.m m.0H m.m z HeOOHmOO mo m mm 2 wave OGOEM comm Hmuoa ocOcmHmHmcosm ocOmouma ocOosoH ocOosoHOmH cowxomasm .cumz + ocOcoOcuoE ocOHm> ocflsma< ocwomaw ocwaoum taco UOEMOSHO soswumm + ocwcoonsa oflom oaunmmmd ocflcflmud osfloflumam osflmmq .O>4 MIHth mlm~m¢ mmIHNM# mm¢ mOHHOflHMNV mowo¢ ocflad mo mfimnuv A.comonpfls swouonmlsos mo madam OH mom mofiom ocOEm .mmoumuom omwuolmnooum mo sowuamomfioo owes ocOEm comm .n manna 55 "the amino acid composition of potato protein is independ- ent of the mineral nutrition of the plants." Further examination of Table 7 reveals that on the average arginine, threonine + serine, and methionine are higher while lysine, aspartic acid and leucine are lower in comparison to the total amino acid values of the same potato varieties. In this study cysteine was not detected in any of the six varieties, and proline was found only in one variety. Threonine and serine appeared as one peak, and their sum is expressed as serine among the free amino acids. This value is unusually high, and, undoubtedly, interfering compounds are present. Nutritionally, the increased value of methionine is important. Methionine is the limiting amino acid in potato; therefore, the contribution of the free methionine to the protein quality of potatoes is significant. The free amino acid nitrogen (ammonia included) was calculated for each of the six varieties, and then compared to the Kjeldahl nitrogen determined in the picric acid supernatant. The recovery of the Kjeldahl nitrogen varied from 26.2% to 54.3%. The soluble N forms mentioned on page 46 may account for this large deficit. The N present in the picric acid non-precipitable fraction was 29% of the total N of the potatoes (Table 1). Of this "soluble" N 37% was recovered in the form of free amino acids found in the picric acid supernatant (Table 7). 56 The free amino acid N, therefore, represents 11% of the total N. Since 80% of the total N was found in the free plus bound amino acids (Table 2), 69% of the total N should be in the form of bound amino acids (proteins and peptides). The remaining 20% N was not accounted for. The free amino acid composition of potatoes re- ported in four investigations and in this study are com- pared in Table 8. Values from the five investigations are not in agreement, a fact which reflects the observations of Mulder and Bakema (1956) that free amino acid composi- tions vary more than amino acids of the protein fraction. Free amino acids take active part in the protein synthesis of the potato, and their composition is thus associated with the rest period and bud formation of the tuber (Cotrufo and Levitt, 1958). Consequently, the seasonal time of free amino acid determination is critical. Estimation of "Total Protein" in Potato by Colorimetric Methods Ninhydrin Method The freeze-dried form of six potato varieties Russet Burbank, #58, #321-65, #322-6, #709 and #711-3 were selected to develop a colorimetric method for the estimation of "total protein" in potatoes. The stable form of the freeze-dried samples provided a model system for the experiment. 57 Table 8. Comparison of the free amino acid composition of potatoes reported by various authors. (Grams of amino acids per 16 grams of non-protein nitrogen.) Chick Thompson Mulder and and and Slack Steward Bakema Hughes This Amino Acids (1949) (1952) (1958) (1958) Study Lysine 1.9 3.3 tr 2.7 4.4 Histidine 1.1 - 5.3 1.4 2.9 Arginine 2.6 31.5 12.0 4.1 10.1 Aspartic acid - 3.1 14.8 28.4 12.9 Threonine 1.1 3.3 2.0 1.7 27.0 Serine - 2.4 3.2 1.1 Glutamic acid - 4.7 33.2 31.1 17.7 Proline - tr tr .3 1.1 Glycine - 1.4 tr .2 .3 Alanine — 5.7 2.6 1.1 2.4 Cystine + Cysteine 1.2 - .9 .6 - Valine 3.3 8.0 8.3 1.7 6.7 Methionine .8 2.1 tr .5 2-1 Isoleucine - - - .5 3.3 Leucine - - - 0 1.7 Tyrosine — ‘ 2.6 6.8 1.6 3.3 Phenylalanine 4.1 3.2 3.6 1.7 3.9 Tryptophan - - - .7 - tr = trace 58 The dye-binding capacities of protein in the freeze-dried samples were tested with orange G dye and later with amido black dye. In the then established ex- perimental conditions none of these dyes gave repeatable, consistent results. Color development by ninhydrin, how- ever, assured reproducible colorimetric readings. Nin- hydrin reacts with the free amino groups of amino acids, peptides and proteins as follows: I 2 .c OH / \ / R—C—COOH'I' 2 C C// \\OH O NH H d-Amino acid Ninhydrin O 0 II II C —N—c/C N co + R—i—O /> \ " 2 I II OH O O Ruhemann's purple Concentration of ninhydrin was best at .015 g per 100 ml. The absorbance of greater or lesser concentration 59 gave absorbance outside of the desired range (Figure l). A sample weight of .50 g was found to be optimal because absorbance started to level off with a greater sample weight (Figure 2). Various methods were tried for the mixing of the sample with ninhydrin. The most consistent results were obtained with a sonifier. The shearing action of the ultra-sound effect of the sonifier violently agitates the contents of the plant cell (Hughes and Nyborg, 1967), and, thus, increases the reaction rate between ninhydrin and the free amino groups in the sample. Good consistency of results was obtained with a shaking time of 1.5 minutes. A shorter mixing time gave poorer result consistency while a longer mixing time decreased the absorbance (Figure 3). Figure 4 demonstrates that the highest absorbance was at- tained when the sample was boiled for 20 minutes after the shaking period. After the optimum conditions were established, the samples of the six potato varieties were tested for nin- hydrin color. The light absorbance of the supernatant of the reaction mixture was measured and plotted against percent "total protein" as % N x 6.25 (Table 9 and Figure 5). A linear regression equation was calculated from the data, and the regression line was drawn accordingly. The regression equation for the correlation of the absorbance of the ninhydrin color with protein content is 60 .800 _ .600 .400 P Absorbance at 570 nm .200 l l l L .010 .015 .020 .025 .030 .035 Concentration of Ninhydrin, g/100 ml Figure 1. Effect of concentration of ninhydrin on color development. .600 .400 t .200 ' Absorbance at 570 nm L l I l 1 l I I .10 .20 .30 .40 .50 .60 .70 .80 Sample weight, grams Figure 2. Effect of potato sample weight on the ninhydrin color development. .600 .400 .200 Absorbance at 570 nm 61 b C) (3 f~ .5 .1.0 1.5 2.0 2.5 Minutes Figure 3. Effect of time of mixing by sonifier on the C O" O O .400 .200 Absorbance at 570 nm color development of ninhydrin with freeze- dried potatoes. l 1 l L J 10 20 30 40 50 Minutes Figure 4. Effect of time of boiling on the color devel- opment of ninhydrin with freeze-dried potatoes. 62 Table 9. "Total protein" content and ninhydrin color of freeze-dried potatoes. Absorbance Varieties "Total Protein" (%) at 570 nm Russet Burbank 10.6 .582 #58 12.3 .808 #321-65 8.1 .217 #322-6 9.9 .460 #709 9.1 .390 #711-3 11.9 .740 Average 10.3 .533 1.200 , 51.000 ‘3 O l‘ Ln 4:3 .800 - Q) 2 (U '3 o .600 . § .400 - .2oo . 63 l 4 l A J I Figure 5. 8 9 10 ll 12 13 Percent "total protein" (% N x 6.25) Correlation between ninhydrin color and per- centage of "total protein" (% N x 6.25) for freeze-dried potatoes. Correlation coefficient is + 0.9987. Standard error of estimate is 0.09%. 64 Y = -.878 + .137X, where Y is the absorbance of ninhydrin color and X is the percentage of "total protein" (% N x 6.25). Accordingly, a table which relates absorbance (Y) and protein content (X) can be prepared by applying the regression equation. For example, when a measured absorb- ance (Y) is .490, the equation will be .490 = -.878 + .137x. Completing the equation: .490 + .878 = .137x or x = 1368/137 = 10.0% "total protein." Figure 5 shows that an absorbance of .490 is equivalent to 10% protein (% N x 6.25). The regression coefficient is .137, the correlation coefficient + 0.9987 and the standard error of estimate 0.09%. The standard error of estimate is similar to the values reported by Ashworth, §E_al. (1960) and Dolby (1961) for milk and by Moss and Kielsmeier (1967) for meat. Dye-binding Method The ninhydrin method was not applicable to the estimation of protein content in raw potato because the colorimetric readings were not reproducible. Orange G dye was also tried, but the repeatability of results remained poor. Presumably, the shearing action of the sonifier was not strong enough to facilitate consistent reaction rates between the anionic dye and the cationic groups of the nitrogenous materials. As a result of the ineffectiveness of the ninhydrin method and initial difficulty of the orange G method attention was directed toward the 65 intactness of the potato cells. A mechanically operated tissue grinder failed to improve the repeatability of the results. Grinding the samples in mortar added no improve- ment to the method. Finally, however, the hand operation of the tissue grinder corrected the discrepencies. Hand grinding of raw potato samples in a tissue grinder appar— ently assured sufficient and consistent cell disruption to provide reproducible colorimetric readings related to the orange G dye-binding capacity of this commodity. The original dye concentration found most suitable was .02 g of dye per liter. With this concentration the absorbance readings of the dye solution after the reaction with the sample was in the range of 0.2 to 0.8 at the wavelength of 480 nm (Figure 6). The Optimum shaking time was 10 minutes: shorter or longer times decreased the amount of bound dye (Figure 7). A ten minute centrifuga- tion resulted in a clear supernatant which was used for measuring the unbound dye concentration spectrophotomet- rically. The difference in absorbance between the original dye solution and the supernatant of the reaction mixture was taken as a measure of the bound dye. When the optimum conditions were established, 21 varieties of raw potato were analyzed for total protein by Kjeldahl. These varieties, the "total protein" as percentage of dry weight and wet weight, and the corres- ponding absorbances of the bound dye are listed in '66 g 1.000 _ c: O (D V‘ b w .800 - (D U G (U .Q ‘5 .600 r 5 J l 1 L L .01 .02 .03 .04 .05 Concentration of dye, g/liter Figure 6. Effect of dye concentration on the absorbance of orange G dye. .350 P E I: O m V .IJ ‘6 0300 '- (D U C: (U .0 H 0 "1 fl 0250 I“ 5 10 15 Minutes Figure 7. Effect of time of shaking on the absorbance of orange G dye bound by raw potatoes. 67 Table 10. Absorbances were plotted against percentage "total protein" (% N x 6.25) for raw potatoes (Figure 8). The linear regression equation for the correlation of the bound dye with total protein is Y = .105 + .091x where Y is the absorbance of bound dye and X is the per— centage of "total protein." By substituting the approp— riate values in the regression equation, a table may be prepared from which total protein content can be read once the absorbance of bound dye is obtained. For example, when the absorbance of bound dye is .300, the percent "total protein" is: .300 = .105 + .091x or .300 - .105 = .091X, from which x = 2.14%. The regression line in Figure 8 verifies this result. The regression coefficient is .091, the correla- tion coefficient +0.9827, and the standard error of esti- mate .07%. The standard error of estimate is similar to those found with the ninhydrin method in this study and in studies reported by Ashworth, gt_al. (1960) and Dolby (1961) for milk and by Moss and Kielsmeier for meat (1967). 68 (Table 10. Dry matter, "total protein" and absorbance at 480 nm of bound orange G dye in raw potatoes. Total Total Dry Protein Protein Matter (% of (% of Absorbance Varieties (%) dry weight) wet weight) at 480 nm .Abnaki 19.2 13.1 2.51 .328 Bakeking 21.0 12.2 2.57 .333 Cascade 18.9 14.0 2.65 .345 Cobbler 20.0 12.7 2.53 .326 Haig 19.2 15.2 2.92 .379 Iopride 20.2 11.3 2.28 .307 Jewel 20.3 10.5 2.14 .298 Katahdin 19.4 12.9 2.50 .328 Kennebec 18.9 12.6 2.38 .316 B514l-6 24.5 11.1 2.73 .353 Manona 19.3 14.2 2.73 .362 Norchip 21.8 14.0 3.04 .373 Pontiac 15.6 16.7 2.60 .335 Russet Burbank 21.3 10.4 2.22 .312 Shurchip 24.8 12.4 3.08 .385 Sioux 18.8 15.9 2.99 .385 Superior 17.3 16.4 2.84 .373 #58 20.9 8.4 1.76 .268 #321-65 27.9 8.9 2.49 .330 #709 14.5 13.0 1.88 .283 #711-3 19.5 11.2 2.19 .307 Average 20.2 12.7 2.53 .335 .400 . .350 .300 Absorbance at 480 nm .250 Figure 8. 69 A J l 2.0 2.5 3.0 Percent "total protein" (% N x 6.25) Correlation between absorbance of bound orange G dye and percentage of "total protein" for raw potatoes. Correlation coefficient is + 0.9827. Standard error of estimate is 0.07%. SUMMARY Evaluation of the Potato Protein by Amino Acid Analysis The quality of proteins in six varieties of potatoes (Solanum tuberosum L.) was evaluated by amino acid analysis. Random samples of the six varieties, Russet Burbank, #58, #321-65, #322-6, #709 and #711-3, were freeze-dried and their content analyzed for nitrogen, "total protein," total sulfur and total and free amino acids. The potatoes analyzed contained from 1.29 to 1.96% N on a dry weight basis corresponding to a "total pro- tein" content (% N x 6.25) of 8.1 to 12.3%. The 10% TCA precipitated protein varied from 30 to 54% of the total protein. The average amino acid composition of the six varie- ties of potato, determined by the Beckman Model 120C Amino Acid Analyzer and reported as g per 16 g N, was: lysine 6.2, histidine 2.3, arginine 4.9, aspartic acid 24.5, threonine 3.8, serine 4.4, glutamic acid 15.5, proline 3.8, glycine 3.0, alanine 3.1, cystine + cysteine 1.3, valine 5.7, methionine 1.6, isoleucine 70 71 4.2, leucine 6.0, tyrosine 3.7, phenylalanine 4.5 and tryptophan 1.5. The individual amino acid concentration in the six varieties varied from amino acid to amino acid. Serine showed the greatest deviation from the average (130%) and arginine the smallest (i4%). Methionine and cystine + cysteine deviated by :12.5 and i15.0% from the six variety average, respectively. The average for the recovery of the Kjeldahl nitrogen as total amino acid N was 80% for the six varieties. The FAQ/WHO (1965) protein scores for the potato varieties Russet Burbank, #58, #321-65, #322-6, #709 and #711-3 were 73, 78, 60, 62, 73, and 68, respect- ively. The average of these protein scores was 69, a value in good agreement with the FAQ/WHO (1965) report which gave a protein score of 70 for the potato. Further comparison revealed that based on protein scores the quality of the potato protein is lower than that of egg and meat, but higher than that of milk, wheat and beans. Protein scores verified that methionine is the limiting amino acid in potato. The free amino acid composition of the six potato varieties was determined by the Technicon Amino Acid Analyzer. The amino acid analysis showed considerable variations in the levels of free amino acids among the six varieties. Also, there was no simple quantitative 72 relationship between the total amino acids and the free amino acids of the potato. The average methionine content was 2.1 g per 16 g N for the free amino acids and 1.6 g per 16 g N for the total amino acids. The average for the recovery of the Kjeldahl nitrogen as free amino acids N was 37% for the six varieties. On the average, 11% of the total N of potatoes was present in the form of free amino acids, 69% in the form of bound amino acids and 20% was unaccounted. Estimation of "Total Protein" in Potato by_Colorimetric Methods Colorimetric methods were developed for rapid estimation of "total protein" in potatoes. 1. The conventional ninhydrin reaction color provided good correlation with percent "total protein" (% N x 6.25) in freeze-dried potatoes. The regression equa- tion Y = -.878 + .137 X was obtained where Y was ab- sorbance of ninhydrin color and X was the percent "total protein" (% N x 6.25). The correlation coeffi- cient was + 0.9987, and the standard error of estimate was 0.09%. Dye-binding with orange G dye gave good correlation to percent "total protein" (% N x 6.25) in raw potatoes. The regression equation Y = .105 + .091 X was derived, where Y was the absorbance of bound dye and X was the 73 percent "total protein" (% N x 6.25). The correlation coefficient was + 0.9827, and the standard of error of estimate was 0.07%. REFERENCES REFERENCES .Agren, G. 1949. Microbiological determination of amino acids in foodstuffs. Acta Chem. Scand. 3, 931- 938. .A1banese, A. A. 1959. Criteria of protein nutrition. In "Protein and Amino Acid Nutrition," ed. Albanese, A. A., pp. 297-347. Academic Press. .Allison, J. B. 1958. In "Protein Nutrition." Ann. N.Y. .Allaway, W. H. and Thompson, J. F. 1966. Sulfur in the nutrition of plants and animals. Soil Sci. 101, 240-247. Ashworth, U. S. 1971. Proteins in meat and egg products determined by dye binding. J. Food Sci. 36, 509- 510. Ashworth, U. S. and Chaudry, M. A. 1962. Dye-binding capacity of milk proteins for Amido Black 10B and Orange G. J. Dairy Sci. 45, 952-957. Ashworth, U. S. and Seals, R. 1958. Factors affecting the dye binding of milk proteins. J. Dairy Sci. 41, 227. Ashworth, U. S., Seals, R. and Erb, R. E. 1960. An im- proved procedure for the determination of milk proteins by dye binding. J. Dairy Sci. 43, 614- 623. Association of Official Analytical Chemists. 1970. "Official Methods of Analysis," 11th Ed. Wash- ington, D.C. Association of Official Analytical Chemists. 1967. Rapid test for protein in milk. J. Assoc. Off. Anal. Chem. 50, 131, 204. Blackburn, S. 1968. "Amino Acid Determination," pp. 1-9, 125-137. Marcel Dekker, Inc., New York. 74 75 Block, R. J. 1960. Amino acid analysis of protein hy- drolysates. In "Analytical Methods of Protein Chemistry," ed. Alexander, P. and Block, R. J., Vol. II. pp. 3-57. Pergamon Press, New York. Block, R. J. and Bolling, D. 1951. "The Amino Acid Com- position of Proteins and Foods," 2nd Ed., pp. 126- 135. Charles C. Thomas, Springfield, Ill. Block, R. J. and Mitchell, H. H. 1946. The correlation of the amino-acid composition of proteins with their nutritive value. Nutr. Abst. and Rev. 16, 249-278. Bontscheff, N. 1965. Uber die Veranderungen im Eiweiss- und Aminosaurengehalt einiger Lebensmittel bei Warmebehandlung und Einwirkung von Enzymen. Nahrung 9(2), 161-166. Borgstrom, G. 1964. The human biosphere and its biolog- ical and chemical limitations. In "Global Impacts of Applied Microbiology," ed. Starr, M. P., pp. 130-163. John Wiley & Sons, Inc., New York. Borgstrom, G. 1967. "The Hungry Planet: The Modern World at the Edge of Famine," pp. 40-41. Collier Books, Collier-Macmillan Ltd., London. Borgstrom, G. 1969. "Too Many: A Study of Earth's Bioloqical Limitations," pp. 42-45. The Macmillan Company, New York. Bressani, R. and Elias, L. G. 1968. Processed vegetable protein mixtures for human consumption in develop- ing countries. In "Advances in Food Research," ed. Chichester, C. O., Mrak, E. M. and Stewart, G. F., 16, 1-103. Academic Press, New York. Bressani, R. and Scrimshaw, N. S. 1961. The development of INCAP vegetable mixtures. I. Basic animal studies. In "Progress in Meeting Protein Needs of Infants and Preschool Children," pp._35-48. Natl. Acad. of Sci.-Nat1. Res. Counc. Publ. 843. Chapman, L. M., Greenberg, D. M. and Schmidt, C. L. A. 1927. Studies on the nature of the combination between certain acid dyes and proteins. J. Biol. Chem. 72, 707-729., Chick, H. and Cutting, M. E. M. 1943. Nutritive value of the nitrogenous substances in the potato. Lancet 76 (flmick, H. and Slack, E. B. 1949. Distribution and nutri- tive value of the nitrogenous substances in the potato., Biochem. J.- 45, 211-221. Clark, J. M. 1964. "Experimental Biochemistry," p. 95. W. H. Freeman and Co., San Francisco. Clark, B. F. C. and Marcker, K. A. 1968. How proteins start. Sci. Am. 218, 36-42. Cotrufo, C. and Levitt, J. 1958. Investigations of the cytOplasmic particulates and proteins of potato tubers VI. Nitrogen changes associated with emergence of potato tubers from the rest period. Physiol. Plant. II, 240—248. de Groot, A. P. and van Stratum, P. G. C. 1963. Biolog- ical evaluation of legume proteins in combination with other plant protein sources. Qual. Plant. Mater. Veg. 10, 168-184. Dolby, R. M. 1961. Dye-binding methods for estimation of protein in milk. J. Dairy Res. 28, 43-55. Dustin, J. P., Czajkowska, C., Moore, S. and Bigwood, E. J. A study of the chromatographic determination of amino acids in the presence of large amounts of carbohydrate. Anal. Chim. Acta 9, 256-262. Dyer, H. J. and Nyborg, W. L. 1960. Ultrasonically- induced movements in cells and cell models. IRE Trans. Med. Electron. 7, 163-165. Finkelstein, J. D. 1970. Control of sulfur metabolism in mammals. In "Symposium: Sulfur in Nutrition," ed. Muth, O. H. and Oldfield, J. E., pp. 46-60. AVI Publishing Co., Westport, Conn. Fithatrick, T. J., Talley, E. A., Porter, W. L. and Murphy, H. J. 1964. Chemical composition of potatoes. III. Relationships between specific gravity and the nitrogenous constituents. Amer. Food and Agriculture Organization of the United Nations. 1957. Protein requirements. FAO Nutritional Studies No. 16, Rome. Food and Agricultural Organization/World Health Organiza- tion Expert Group. 1965. Protein requirements. World Health Organ. Tech. Rept. Ser. 301, Geneva. 77 Fraenkel-Conrat, H. and Cooper, M. 1944. The use of dyes for the determination of acid and basic groups in proteins. J. Biol. Chem. 154, 239-246. Garrigus, U. S. 1970. The need for sulfur in the diet of ruminants. In "Symposium: Sulfur in Nutrition," ed. Muth, O. H. and Oldfield, J. E., pp. 126-152. AVI Publishing Co., Westport, Conn. Gaunt, S. N. and Gacula, Jr., M. C. 1964. Effect of time on Orange G dye protein determinations. J. Dairy Sci. 47, 712. Gaunt, S. N. and Hankinson, D. J., 1969. Calibration techniques for protein estimation by the acid Orange 12 dye binding method. J. Dairy Sci. 52, 278. Groot, E. H. 1946. Investigation into the biologically important amino acids in potato protein, in con- nection with its nutritive value, I-V. Arch. neerl. de Physiol., 28, 277-361. Habib, A. T. and Brown, H. D. 1956. Factors influencing the color of potato chips. Food Technol. 10, 332-336. Hegsted, D. M. 1964. Proteins. In "Nutrition: A Com- prehensive Treatise," ed. Beaton, G. H. and McHenry, E. W., Vol. I, pp. 116-117. Academic Press, New York. Hellendoorn, E. W. 1969. Intestinal effects following ingestion of beans. Food Technol. 23, 795-800. Hirs, C. H. W. 1956. The oxidation of ribonuclease with performic acid. J. Biol. Chem. 219, 611-621. Hirs, C. H. W., Stein, W. H. and Moore, S. 1954. The amino acid composition of ribonuclease. J. Biol. Hirsch, J. S., Niles, A. D. and Kemmerer, A. R. 1952. The essential amino acid content of several vege- tables. Food Research 17, 442-447. Hughes, B. P. 1958. The amino-acid composition of potato protein and of cooked potato. British J. Nutr. 12, 188-195. 78 Jaschik, S. és Lindner, K. 1955. Adatok a kfilSanzB talajokon termelt étkezési burgonyék tépanyag osszetételéhez. NBVénytermelés 4, 213-217. Kellogg, J. H. The special dietetic virtues of the potato. Quoted in Grubb, E. H. and Guilford, W. S. 1912. "The Potato," pp. 7-16. Doubleday, Page and Co., Garden City, New York. Kjeldahl, J. 1883. Neue Methode zur Bestimmung des Stickstoffs in organishen KBrpern. Z. anal. Chem. Kofranyi, E. und Jekat, F. 1965. Die biologische Wertigkeit von Kartoffelproteinen. Forschber Landes NRhein-Westf., Nr. 1582, I. Kon, S. K. 1928. The nutritional value of tuberin, the globulin of potato. Biochem. J. 22, 261-267. Kon, S. K. and Klein, A. 1928. The value of whole potato in human nutrition. Biochem. J.' 22, 258-260. Lampitt, L. H. and Goldenberg, N. 1940. The composition of the potato. Chemistry and Industry. 18, 748- 761. Langer, W. L. 1963. Europe's initial population explo- sion. Am. Historical Review. 69, 1-17. Lehninger, A. L. 1970. "Biochemistry: The Molecular Basis of Cell Structure and Function," pp. 698- 700. Worth Publishers, Inc., New York. Lewis, O. A. M. 1966. Short ion-exchange column method for the estimation of cystine and methionine. Nature. 209, 1239-1240. Lindner, K., Jaschik, S. und Korpaczy, I. 1960. Aminosaurenzusammensetzung und Biologischer Wert der Kartoffeleiweissfraktionen. Qual. Plant. Mater. Veg. 7, 289-294. Lindner, K., Jaschik, S., Korpaczy, I., Polner,.R. és Vardi, P. 1954. A magyar burgonya taplalkozési értéke. N6vénytermelés 3, 261-280. Loeb, J. 1924. "Proteins and the Theory of Colloidal Behavior," 2nd Ed., p. 36. Quoted in Fraenkel- Conrat, H. and Cooper, M. 1944. The use of dyes for the determination of acid and basic groups in proteins. J. Biol. Chem. 154, 239-248. 79 Lyman, C. M. and Kuiken, K. A. 1949. The amino acid composition of meat and some other foods. Texas Agr. Expt. Sta. Bull. 708. McCarthy, T. E. and Sullivan, M. X. 1941. A new and highly specific colorimetric test for methionine. J. Biol. Chem. 141, 871-876. .Miller, D. S. and Donoso, G. 1963. Relationship between the sulfur/nitrogen ratio and the protein value of diets. J. Sci. Food Agr. 14, 345-349. Miller, D. S. and Naismith, D. J. 1958. A correlation between sulphur content and net dietary-protein value. Nature 182, 1786-1787. Mitchell, H. H. 1924a. The biological value of proteins at different levels of intake. J. Biol. Chem. 58, 905-922. Mitchell, H. H. 1924b. A method of determining the bio- logical value of protein. J. Biol. Chem. 58, 873-903. Mitchell, H. H. 1924c. The supplementary relations among proteins. J. Biol. Chem. 58, 923-929. Mitchell, H. H. and Block, R. J. 1946. Some relationships between the amino acid contents of proteins and their nutritive values for the rat. J. Biol. Chem. 163, 599-620. Mohler, H. und Sulser, H. 1968. Kartoffeln und Kartoffel- Erzeugnisse. In "Handbuch der Lebensmittelchemie," Gesamtred. Schormfiller, J., Band V/2 Teil, S. 473-506. Springer-Verlag, Berlin-New York. Moore, 8., Spackman, D. H. and Stein, W. H. 1958. Chromatography of amino acids on sulfonated poly- styrene resins. Anal. Chem. 30, 1185-1190. Moore, S. and Stein, W. H. 1948. Photometric ninhydrin method for use in the chromatography of amino acids. J. Biol. Chem. 176, 367-388. Moore, S. and Stein, W. H. 1951. Chromatography of amino acids on sulfonated polystyrene resins. J. Biol. Chem. 192, 663-681. 80 Moore, S. and Stein, W. H. 1954. Procedures for the chromatographic determination of amino acids on four percent cross-linked sulfonated polystyrene resins. J. Biol. Chem. 211, 893-906. Moss, V. G. and Kielsmeier, E. W. 1967. A method for rapid determination of protein in meat by dye binding. Food Technol. 21, 351-354. Mulder, E. G. and Bakema, K. 1956. Effect of the nitro- gen, phosphorus, potassium and magnesium nutrition of potato plants on the content of free amino- acids and on the amino-acid composition of the protein of the tubers. Plant and Soil. 7, 135- 166. Neuberger, A. and Sanger, F. 1942. The nitrogen of the potato. Biochem. J. 36, 662-671. Nielsen, E. V. 1970. NPR-fertilizer, the influence of nitrogen-, phosphorus- and potassium supply on: (1) yield of tubers and dry matter; (2) concentra- tion of some elements in the tuber. Proc. Fourth Triennial Conf. of the EurOpean Assoc. for Potato Res. I.N.R.A. Publ. 70-5, pp. 159-160. EurOpean Assoc. for Potato Res., Wageningen (Netherlands). Orr, M. L. and Watt, B. K. 1957. Amino acid content of foods. Home Economics Res. Rep. No. 4. U.S.D.A. Washington, D.C. Osborne, T. B. and Mendel, L. B. 1914. Amino-acids in nutrition and growth. J. Biol. Chem. 17, 325-349. Pyke, M. 1970. "Man and Food," pp. 134-146. McGraw-Hill Book Co., New York. Rawlins, L. M. C. and Schmidt, C. L. A. 1929. Studies on the combination between certain basic dyes and proteins. J. Biol. Chem. 82, 709-716. Rawlins, L. M. C. and Schmidt, C. L. A. 1930. The mode of combination between certain dyes and gelatin granules. J. Biol. Chem. 88, 271-284. Rhee, K. C. 1969. Isolation and characterization of lacteal immunoglobulins. Ph.D. thesis, Michigan State University, East Lansing, Michigan. Rose, W. C. 1949. Amino acid requirements of man. Fed- eration Proc. 8, 546-552. 81 Rose, W. C., Haines, W. J. and Warner, D. T. 1954. The amino acid requirements of man. V. The role of lysine, arginine, and tryptophan. J. Biol. Chem. 206, 421-430. Rose, W. C., Oesterling, J. M. and Womack, M. 1948. Comparative growth on diets containing ten and nineteen amino acids, with further observations upon the role of glutamic and aspartic acids. J. Biol. Chem. 176, 753-762. Rose, W. C. and Wixom, R. L. 1955. The amino acid re- quirements of man. XIII. The sparing effect of cystine on the methionine requirement. J. Biol. Chem. 216, 763-773. Schram, E., Dustin, J. P., Moore, S. and Bigwood, E. J. 1953. Application de la chromatographie sur échangeur d'ions a l'étude de la composition des aliments en acides amines. Anal Chim. Acta. 9, 149-162. Schram, E., Moore, 8. and Bigwood, E. J. 1954. Chromato- graphic determination of cystine as cysteic acid. Schuphan, W. 1958. Differences génétiques de qualité des protéines et de teneurs en amino-acides indispen- sables chez les végétaux alimentaires. Qual. Plant. Mater. Veg. 3-4, 34-44. Schuphan, W. 1960a. Studien fiber essentielle Aminosauren in Kartoffeln. 2. Mitt. Die biologische Eiweisswertigkeit der Kartoffel (Solanum tuberosum L.) im Ernahrungsversuch und im SpiegeI der essentiellen Aminosauren. Qual. Plant. Mater._ Veg. 6, 16-38. Schuphan, W. 1960b. Die Biologische Eiweisswertigkeit der Kartoffel als Qualitatsmasstab ffir Sorten- bewertung und optimale Dfingung. Qual. Plant. Mater. Veg. 6, 293-297. Schuphan, W., Mfihlendyck, E. und Overbeck, G. 1969. Wertgebende Inhaltsstoffe der Kartoffel in Abhangigkeit von verschiedenen haushaltsmassigen Zubereitungen. Mitt. I. Allgemeines, Unter- suchungsmaterial und Methodik. Qual. Plant. Mater. Veg. 17, 169-178. 82 Schuphan, W. et Postel, W. 1958. Action de la fumure et du climat sur la teneur en amino-acides indis- pensables. Qual. Plant. Mater. Veg. 3-4, 45-61. Schwerdtfeger, E. 1969. Wertgebende Inhaltsstoffe der Kartoffel in Abhangigkeit von verschiedenen haushaltsmassigen Zubereitungen. Mitt. 3. Eiweiss und Aminosauren. Qual. Plant. Mater. Veg. 17, 191-200. Seals, R. G., Nissen, C., Burrow,.C. D. and Hammond, E. G. 1969. Some possible sources of error in dye bind- ing methods. J. Dairy Sci. 52, 881. Sheffner, A. L. 1967. In vitro protein evaluation. In "Newer Methods of Nutritional Biochemistry," ed. Albanese, A. A., Vol. III, pp. 125-195. Academic Press, New York. Sheffner, A. L., Eckfeldt, G. A. and Spector, H. 1956. The pepsin-digest-residue (PDR) amino acid index of net protein utilization. J. Nutr. 60, 105-120. Sherbon, J. W. 1967. Rapid determination of protein in milk by dye binding. J. Assoc. Off. Anal. Chem. 50, 542-547. Sherbon, J. W. and Hemphill, B. 1967. Comparison of the reproducibilities of the Kjeldahl and dye binding methods for measuring protein in milk. J. Assoc. Off. Anal. Chem. 50, 557-560. Sjollema, B. und Rinkes, I. J. 1912. Die Hydrolyse des Kartoffeleiweisses. Physiologische Chemie 76, 369-384. Slack, E. B. 1948. Nitrogenous constituents of the potato. Nature 161, 211-212. Smith, 0. 1968. Chemical composition of the potato. In "Potatoes: Production, Storing, Processing," ed. Smith, 0., pp. 59-109. AVI Publishing Co., West- port, Conn. Spackman, D. H., Stein, W. H. and Moore, S. 1958. Auto- matic recording apparatus for use in the chroma- tography of amino acids. Anal. Chem. 30, 1190- 1206. Spies, J. R. 1967. Determination of tryptophan in pro- teins. Anal. Chem. 39, 1412-1416. 83 Spies, J. R. and Chambers, D. C. 1948. Chemical deter- mination of tryptophan. Anal. Chem. 20, 30-39. Steel, R. G. D. and Torrie, J. H. 1960. "Principles and Procedures of Statistics," pp. 161-193. McGraw- Hill Book Co., Inc., New York. Steinholt, K. 1957. A colorimetric method for the quan- titative determination of protein in milk. Meieriposten, 46, 901-905. (Dairy Sci. Abstr., 20, 161. 1958.) Stewart, B. A. and Porter, L. K. 1969. Nitrogen-sulfur relationships in wheat (Triticum aestivum L.), corn (Egg ma 3), and beans (Phaseolus vulgaris). Agron. J. , 267-271. Sweeney, J. P., Hepner, P. A. and Libeck, S. Y. 1969. Organic acid, amino acid, and ascorbic acid con- tent of potatoes as affected by storage conditions. Amer. Potato J. 46, 463-469. Talley, E. A., Fithatrick, T. J. and Porter, W. L. 1961. Chemical composition of potatoes. I. Preliminary studies on the relationships between specific gravity and the nitrogenous constituents. J. Food Sci. 26, 351-355. Talley, E. A., Fithatrick, T. J. and Porter, W. L. 1964. Chemical composition of potatoes. IV. Relationship of the free amino acid concentrations to specific gravity and storage time. Amer. Potato J. 41, 357-366. Talley, E. A., Fithatrick, T. J. and Porter, W. L. 1970. Chemical composition of potatoes. VIII. Effect of variety, location, and year of growth on the con- tent of nitrogen compounds. Amer. Potato J. 47, 231-244. Talley, E. A. and Porter, W. L. 1970. Chemical composi- tion of potatoes. VII. Relationship of the free amino acid concentrations to Specific gravity and storage time. Amer. Potato J. 47, 214-224. Thomas, K. 1909. Uber die biologische Wertigkeit der Stickstoffsubstanzen in verschiedenen Nahrungsmitteln. Beitrage zur Frage nach dem physiologischen Stickstoffminimum. Arch. Anat. Physiol. - , 219-305. 84 Thompson, J. F., Morris, C. J. and Gering, R. K. 1960. The effect of mineral supply upon the amino acid composition of plants. Qual. Plant. Mater. Veg. 6, 261-275. Thompson, J. F., Smith, I. K. and Moore, D. P. 1970. Sulfur requirement and metabolism in plants. In "Symposium: Sulfur in Nutrition," ed. Muth, O. H. and Oldfield, J. E., pp. 80-96. AVI Publishing Co., Westport, Conn. Thompson, J. F. and Steward, F. C. 1952. The analysis of the alcohol-insoluble nitrogen of plants by quan- titative procedures based on paper chromatography. II. The composition of the alcohol-soluble and insoluble fractions of the potato tuber. J. Exptl. Botany 3, 170, 181-187. Toennies,.G. 1942. The oxidative conversion of casein into protein free of methionine and tryptOphan. Toennies, G. and Homiller, R. P. 1942. The oxidation of amino acids by hydrogen peroxide in formic acid. Toepfer, H. 1965. "Instruction Manual. Model 120C Amino Acid Analyzer." Spinco Division, Beckman Instru- ments, Inc., Palo Alto, California. Udy, D. C. 1954. Dye-binding capacities of wheat flour protein fractions. Cereal Chem. 31, 389-395. Udy, D. C. 1956a. Estimation of protein in wheat and flour by ion-binding. Cereal Chem. 33, 190-197. Udy, D. C. 1956b. A rapid method for estimating total protein in milk. Nature 178, 314-315. Wertz, A. W., Ruttenberg, P. K., French, G. P., Murphy, G. H. and Guild, L. P. 1956. Amino acid content of foods. J. Am. Dietetic Assoc. 32, 926-928. Westerlind, E. 1970. The potato as a source of protein. Factors affecting yield and content of protein and its amino acid composition. Proc. Fourth Triennial Conf. of the European Assoc. for Potato Res. I.N.R.A. Publ. 70-5, pp. 174-175. European Assoc. for Potato Res., Wageningen (Netherlands). ‘White, A., Handler, P. and Smith, E. L. 1968. "Principles of Biochemistry," 4th Ed., pp. 582-583. McGraw- Hill Book Co., New York. Y LIBRARIES Hmmum 6142 "11111111111111“WWW 3 1293 03111