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"g . , -" 00-7-1 . odgooo-aQfirr‘.q' C'. “MI-D _.__._ . ‘— l 5 ‘ L 13 R A R Y .- % Michigan Staff: 1 University ABSTRACT EVALUATION OF METHODS TO DETERMINE THE SULFUR CONTAINING AMINO ACIDS IN POTATOES BY Robert Luescher The methionine content of six potato varieties were estimated by Leuconostoc mesenteroides P-60 with acid hydrolysis and by Streptococcus zymgggnes using both acid and enzymatic hydrolysis techniques. By applying the same assay structure, the three methods could becompared for accuracy, recovery and economics. All methods differ- Ientiated high, medium, and low methionine contents. Values received from L. mesenteroides were approximately 30% lower than those obtained by S. gymogenes and acid hydrolysis. With two exceptions the latter ones agreed with the results obtained through ion exchange chromatography (Kaldy, 1971). The technique using 8. zymogenes and enzymatic diges- tion was found to be most appropriate for analyses of a large number of samples since the procedure was least time consuming and the results accurate enough to differentiate among high, medium and low methionine contents. This method permits measurement of the "available" methionine which may correspond more closely with the results in "vivo." Cystine analysis for the same cultivars was conducted with'L.'mesenteroides. Values obtained were about half of those from ion exchange chromatography. A definite explana- tion of this phenomena could not be given. Robert Luescher In further assays ten randomly chosen cultivars from each of 18 families were analyzed for methionine using 5. zymoggnes and enzymatic hydrolysis. From the variance of the population mean, 43% could be explained by differ- ences within families, 55% by differences among families and 2% by variance due to the method. Methionine varied over a range of 1.0 up to 3.6 ug l-met per mg d.m. EVALUATION OF METHODS TO DETERMINE THE SULFUR CONTAINING AMINO ACIDS IN POTATOES By Robert Luescher A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Crop and Soil Sciences 1971 ACKNOWLEDGMENTS The author would like to express appreciation and thanks to Dr. N. R. Thompson for his stimulating interest and his very liberal thoughts which encouraged this study to a great extent. Sincere appreciation is also expressed to Dr. R. J. Evans and Miss D. H. Bauer whose technical assistance was invaluable at the initiation of this study. Gratitude is expressed to Dr. D. Penner for his cooperation throughout this research. ii TABLE OF CONTENTS Page INTRODUCTION . . . . . . . . . . . . . . . . . . . . . 1 LITERATURE REVIEW . . . . . . . . . . . . . . . . . Method to determine methionine . . . . . . . Advantages and disadvantages of ion exchange chromatography . . . . . . . . . . . . . Advantages and disadvantages of paper chromatography . . . . . Advantages and disadvantages of microbiological methods . . . . . . . . . . . . . . . . . . . . Choice of appropriate microorganisms . . . . . m-bkblmw \l MATERIALS AND METHODS O O O O O C C O C O O C O O O O 1. Methods and characteristics of S. zymogenes . 7 a. Procedure for "available" meth1onine . . . 8 b. Procedure for "total" methionine . . . . 11 II. Method using Leuconostoc mesenteroides P- 60 for "total" methionine . . . . . 12 III. Method using Leuconostoc mesenteroides P-60 for "total" cystine . . . . . . . . . . . . 13 IV. Materials and preparations of potato samples . 14 RESULTS AND DISCUSSION . . . . . . . . . . . . . . . . 16 Methionine analysis of 6-cultivars conducted with'L.'mesenterOides'P460, acid hydrolysis . 17 Methionine analysis of 6 cultivars condutted with S. zymogenes, acid hydrolysis . . . . . . 21 Methionine analysis of 6 cultivars using S. zYmgggnes, and enzymatic digestion . . . . . . 24 Discu551on and conclusions of methods evaluating methionine . . . . . . . . . . . . . 33 Cystine analysis conducted by using L. mesenterOides P- 60, acid hydrolysis . . . . . . 35 Methionine analysis of 180 potato clones con- ducted by using 8. zymogenes,enzymatic digestion . . . . . . . . . . . . . . . . . . . 40 iii TABLE OF CONTENTS (Cont.) Page REFERENCES . . . . . . . . . . . . . . . . . . . . . . 49 Appendix 1: Basal medium for S. zymogenes . . . . . . 54 Appendix 2: Amino acid supplement for S. zymogenes . 55 Appendix 3: Citrate cyanide buffer . . . . . . . . . 56 Appendix 4: Basal medium and amino acid supplement for L. mesenteroides,met assay . . . . . 57 Appendix 5: Composition of the cystine assay medium for L. mesenteroides P-60 . . . . . . . . 59 iv 10. 11. 12. LIST OF TABLES Bacteria and the amino acids for which they have a specific requirement . . . . . . . . . . Analysis of variance of methionine conducted with L. mesenteroides P-60, acid hydrolysis Recovery control of methionine . . . . . . Analysis of variance of methionine content estimated by means of S. zymogenes, acid hYdrOIYSiS O O O O O O O O I O O O O O O I Recovery control of methionine . . . . . . Procedure to correct basic turbidity . . . . . Analysis of variance of methionine content estimated with S. zymoggnes, enzymatic digestion . . . . . . . . . . . . . . . . . Recovery control of methionine . . . . . . . . . Summarized results, part I . . . . . . . . Summarized results, part II . . . . . . . . Analysis of variance of cystine estimated by using L. mesenteroides P-60, acid hydrolysis . Recovery control of cystine . . . . . . . . . . Page 17 19 21 23 26 27 28 32 33 37 LIST OF FIGURES Page Hierarchic assay structure . . . . . . . . . . . . 16 Absorbance curve of uninoculated and inoculated potato starch samples partially supplemented with methionine . . . . . . . . . . . . . . . . 25 Cultivar means of methionine of all methods . . . 30 Cultivar means of cystine . . . . . . . . . . . . 31 Frequency distribution of methionine of 180 Clones I 0 O O O O O O O 0 O O O O I O O O O O 0 45 Frequency distributions of methionine of 180 clones according to families . . . . . . . . . . 46 vi INTRODUCTION Among the world's most important food crops, the potato ranks firSt in terms of volume of fresh product. While usually classed as a carbohydrate, it has been in most cases by-passed as a source of protein. In its fresh state, it has only an average of 2% "total protein." How- ever, on a dry basis the "total protein" content of pota- toes is not different from that of wheat. Also, an impor- tant fact overlooked by many is that one hectare of land under potato cultivation can supply the protein requirement for 9.5 people, while the protein of wheat from the same land can satisfy only 6.3 people (Borgstrom, 1969). More- over, the biological value for the potato for the human adult is 72 compared to 53 for wheat flour (FAO, 1957). Investigations have shown (Watts 33 31., 1959; Williams, 1959) that the delivery of methionine for human nutrition is a pathway and it appears that this amino acid limits the biological value of potato protein (Williams, 1959; Schuhpan, 1958; Rios, 1969). Therefore, it would be very desirable to develop a potato with an increased methionine content. To achieve this aim, the potato breeder needs simple methods to screen large numbers of potato clones for methionine. 1 2 The purpose of this thesis is to find and adapt an appropriate method for the above mentioned purpose. This method must be fairly accurate, rapid, and economical. Since cystine may substitute in the diet for about 30% of the methionine (Albanese and Orto, 1968), an appropriate method for the analysis of cystine would be desirable. LITERATURE REVIEW Method to determine methionine There are good methods for the determination of some amino acids, for instance tryptophan (Spies and Chambers, 1948; Spies, 1967). The sulfur containing amino acids are the most difficult to determine. Both methionine and cystine are very unstable during acid hydrolysis, especially in the presence of carbohydrates (Block, 1956 a). At present there are no rapid or simple methods available for deter- mining methionine and cystine. Advantages and disadvantages of ion exchange chromatography The introduction of an automatic chromatographic separation procedure for amino acids (Spackman gt 21., 1956) opened a new area in this field. Spackman 35 31. (1956) reported an accuracy of 100 t 3% and in less than ten hours an analysis of almost all amino acids could be performed. Some difficulties were reported in the recovery of methionine and cystine (Block, 1956 a). The same problem was observed when the same samples were analyzed by Kaldy (1971) and in this study. 4 A special oxidation of cystine and cysteine to cysteic acid and methionine (Schram 2: 21., 1954; Lewis, 1966) to methionine sulfone was necessary in order to get satis- fying results for these amino acids. Furthermore the amino acid tryptophan must still be determined in a separate analysis by a special method (Spies and Chambers, 1948; Spies, 1967). Since the protein has to be broken down to its amino acids by means of acid or alkaline hydrolysis, the "total" amino acids are determined by chromatographic methods. Furthermore the cost for a total analysis is too high to be practical for screening segregating populations in a heterozygous tetraploid. Advantages and disadvantages of_paper chromatography Since paper chromatography requires simple equipment, this procedure is much less expensive than ion exchange chromatography. But the quantitative determination of single amino acids is quite laborious and the accuracy is only about 100 i 6% (Mulder and Bakema, 1956). Furthermore acid hydrolysis and the removal of the acid (HCl) is re- quired for both paper chromatography (Mulder and Bakema, 1956) and for ion exchange chromatography (Spackman 33 31., 1956). Advantages and disadvantages of microbiological methods All microbiological assay methods in current use are similar and can be applied with equal facility to any amino 5 acid for which techniques have been established. The value, especially for routine analysis, is readily apparent (Block, 1956 b). The growth of living cells is subject to many more extraneous influences than are in-vitro chemical reactions or physical measurements (Kidder and Dervey, I949). The most important advantages of microbioassays for amino acid analyses are that there are highly specific bacteria - and simple techniques applicable for all amino acids. The methods are well adapted to routine work (Guggenheim, 1970). Choice of appropriate microorganisms Only L. mesenteroides and S. zymogenes can be used for methionine and cystine analyses (Table l). Table 1: Bacteria and the amino acids for which they have a specific requirement. L. arabinosus L. helveticus L. brevis L. mesenteroides S. faecalis S.<§ymggenes Pediococcus cerevisiae leucine, isoleucine, valine, tryptophan, glutamic acid serine, aspartic acid glycine, proline lysine, phenylalanine, histidine, serine, glycine, proline, aspartic acid, methionine, cystine, glutamic acid, tyrosine, arginine, leucine, isoleucine, valine lysine, hystidine, arginine, threonine methionine, leucine, isoleucine, valine, arginine, histidine, tryptophan, glutamic acid alpha-alanine Source: Based on Barton-Wright (1952) and the newer studies of Ford (1962). MATERIALS AND METHODS I. Methods and characteristics of S. zymogenes The standardized method used in these tests is a modification of Boyne gt 21. (1967). Reagents Assay medium: The assay medium was prepared from two stock solutions, the basal medium and the amino acid supplement, made up as described in Appendix 1 and 2. Stock solutions were stored at -20 C. When required for use, the stock solutions were thawed and the amino acid supplement warmed to dissolve the precipitate. They were then combined in the proportion of two volumes of basal medium to one volume of amino acid supplement. Organism maintenance and inocula Streptococcus zymogenes (NCDO 592) was obtained from the National Culture of Daily Organisms of the National Institute for Research in Dairying. Stock cultures were maintained by weekly (1) transfer (on Bacto Assay Culture Agar B 319, Difco Laboratories, Inc., Detroit, Mich.), incubation over night, and storage at 2 C (2). To prepare the inoculum for the assay, the 8 broth (Bacto Micro Inoculum Broth B 320, Difco Laboratories, Inc., Detroit, Mich.) was inoculated directly from the stock culture (3) and incubated over night at 37 C. Modifications: (1) weekly transfer instead of monthly transfer (2) 2 C instead of 4 C (3) transfer medium was omitted a. Procedure for "available" methionine The "available" methionine content was determined by using S. zymoggnes and enzymatic hydrolysis. For "total" methionine, acid hydrolysis was used instead of enzymatic hydrolysis. Preparation of the samples for tests: The freeze- dried samples (preparation see page 14) were ground to pass an 80 (l) mesh sieve. A half gram (2) t 0.5% was weighed into 4 oz. wide mouth screw cap bottles (in duplicate) and suspended in 20 ml of citrate cyanide buffer (see Appendix 3). The pH was adjusted to 7.2 and the container placed in a water bath at 56 C. Two ml of 4% (W/V) crude papain (Difco Laboratories, Inc., Detroit, Mich.) in citrate cyanide buffer at pH 7.2 was added and incubated with inter- mittent shaking for-3 hr at 56 C. The pH of the digest was adjusted to 7.2. Then, 78 m1 of distilled water was added bringing the volume to 100 m1 1 0.5 ml. 9 Determination of "available" methionine: Triplicate portions of 0, 1, 2, 3, 4, 5, 6, 7, and 8 ml of the standard methionine (10 ug l-met/ml) were distributed into 16 x 150 mm test tubes. Duplicate 2, 3, and 4 m1 portions of each sample were pipetted into the same sized test tubes. Three m1 of assay medium was added to each tube and brought to the final volume of 11 ml with distilled water. Each tube was covered with a test tube cap, sterilized by steaming (100 C) for 20 minutes, and cooled in a water bath to 37 C. Then one drop of inoculum culture that had been diluted 1:10 with 0.85% saline solution (3) was added and the samples incubated at 37 C for 48 hours. Modifications: (l) 80 mesh instead of 40 mesh (2) 0.5 g instead of 100 mg N (3) undiluted inoculum was used in the original paper In this caSe a sample of 0.5 g potato starch was used as a blank. Measurement of growth response: After incubation the tubes were heated in flowing steam for 10 minutes and cooled to room temperature. The tubes were stoppered and shaken very vigorously, then set aside for 30 seconds to allow air bubbles to rise but not food particles to settle. Correc- tion of turbidity caused by food particles is outlined on page 26. The optical densities of the cultures were 10 measured with a HitaEhi Perkin-Elmer 139 UV-VIS spectro- photometer with a flow through cell at 580 um (Ford, 1960). Calculation of results: A curve of the average responses of the standard cultures was plotted. From this curve the values for each sample culture were calcu- lated. Samples with cultures which differed more than 15% of its mean were repeated. Characteristics of S. zymogenes S. zymggenes has an absolute requirement for exo- genous 1eucine*, methionine*, tryptophan*, arginine, histidine, isoleucine*, valine*, and glutamic acid. Of the "essential" amino acids, lysine, threonine, and phenyl- alanine were not absolutely essential to this bacteria though the omission of any one from the culture medium caused a marked drOp in growth rate (Ford, 1960). Furthermore Ford (1960) describes S. zymogenes as follows: "It requires much the same pattern of exogenous amino acids as Tetrahymena: it is powerfully proteolytic, and grows quickly with an adequate intact protein as the main source of nitrogen." Its ability to work as a proteolytic microorganism makes S. zymogenes a very important tool. Instead of chemical hydrolysis, the enzymatic predigestion is * Essential for man. ll satisfactory. Chemical hydrolysis may cause destruction or unavailability of certain amino acids in the food protein, especially when large amounts of carbohydrates are present (Evans and Butts, 1948; Block, 1956 a). Acid hydrolysis can also destroy growth inhibitory substances. Enzymatic digestion permits measurement of the "available" amino acids which may more closely correspond with the results in "vivo." The saving of time by enzymatic digestion compared to the acid hydrolysis is shown later. Unfortunately it is not possible to determine "available" cystine from prepared enzymatic hydrolysates. In this respect L. mesenteroides is a superior microorganism because "total" cystine as well as "total" methionine can be determined from the same acid hydrolysate. b. Procedure for "total" methionine The methionine analyses follow similar procedures except for preparation of the samples for analysis: Dupli- cate 0.5 g samples were weighed into 50 ml Erlenmeyer flasks and hydrolyzed as described by Evans 35,31. (1962): Ten ml of 20% HCl was added to each flask. After auto- claving for 30 minutes at 15 pounds pressure (121 C) the samples were cooled in ice water, the pH adjusted to 7.0 with 12% and 4% NaOH. Then the samples were filtered and brought to a volume of 100 ml with distilled water. 12 11. Method usin Leuconostoc mesenteroides P-60 for "total" met ionine The standardized method was based on that described in the Difco Manual (1969) using dehydrated Bacto-Methionine Assay Medium (Difco Laboratories, Inc., Detroit, Mich.) according to the formula given by Steel 35 El: (1949). Reagents To rehydrate the medium, 105 grams of Bacto Methionine Assay Medium was suspended in 100 ml distilled water and filtered before use. Organism maintenance and inocula L. mesenteroides (ATC 8042) cultures were maintained and test samples prepared as described under S.§ymggenes (pages 7, ll). Triplicate portions of 0, 0.5, l, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, and 5 m1 of the standard methionine (15 ug l-met/ml) were distributed into 18 x 150 mm test tubes. Duplicate 0.4, 0.8, and 1.2 ml portions of each sample were pipetted into the same sized test tubes. Five ml of the prepared Bacto-Methionine Assay Medium was added to each tube and brought to the final volume of 10 ml with distilled water. Each tube was covered with a test tube cap, sterilized in an autoclave at 15 pounds pressure (121 C) for 10 minutes, and cooled in a water bath to 37 C. Then one drop of inoculum culture that had been diluted 1:10 with 0.85% saline solution was added and the samples incubated at 37 C for 24 hours. 13 Measurement of_grpwth response: All steps were per- formed asdescribed for S. zymogenes with the exception of wavelength, which was set at 700 mu for this bacterium (Difco Manual, 1969). Calculation of results were performed as described under S. zympgenes (page 10). III. Method usingLeuconostoc mesenteroides P-60 for "total"’cystine For "total" cystine acid hydrolysis was used. Since L. mesenteroides does not have proteolytic characteristics it barely grows on enzymatic digests. These enzymatic digests are mainly composed of peptides and low molecular proteins which have to be broken down by bacterial enzymes. Only S. zymoggnes has such enzymes. There is no known proteolitic bacteria for which cystine is an essential amino acid. Therefore the determination of "total" cystine in this study was restricted. For cystine the oxidized peptone medium of Lyman pp a1. (1946) in combination with the basal medium made up as recommended by Evans and Bauer (1971) was used. Reagents Composition and preparation see Appendix 5. Preparation of the samples for test: The hydrolysates used for "total" methionine with S. zzmogenes were used for L. mesenteroides. 14 Determination of "total" cystine: Triplicate portions of 0, 0.5, l, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, and 5 m1 of standard cystine (2 ug 1-cystine/m1) were distributed into 18 x 150 mm test tubes. Duplicate 0.4, l, 1.6 m1 portions of each sample were pipetted into the same sized test tubes. Five ml of the prepared cystine assay medium was added to each tube and brought to a final volume of 10 ml with distilled water. Each tube was covered with a test tube cap and sterilized by autoclaving at exactly 15 pounds pressure (121 C) for only 8 minutes. Inoculation, measure- ment of growth response, and calculation of results were performed as previously described under S. zymogenes (page 9). IV. Materials and_preparations of potato samples For the evaluation of methods to determine the sulfur containing amino acids, the following potato clones were chosen: Russet Burbank, Merrimack 58, Michigan State 321-65, 322-6, 709 and 711-3. The potatoes were grown on the Montcalm Experimental Farm in 1969 on a Montcalm sandy loam, fertilized with 128 lbs/A of N, 192 lbs/A of P205 and 192 lbs/A of K20. Eight inches of water was added as irrigation. After harvest the potatoes were stored at 40 F until April 1970. Preparation of samples: From each variety 8 lbs of tubers was randomly chosen, thoroughly washed, and sliced into 1 to 2 mm thick slices and placed immediately on a dry 15 ice layer where they froze completely within 3 to 5 minutes. The samples were kept frozen at -20 C until they could be freeze dried. After freeze drying the samples were ground through an 80 mesh sieve in a Wiley mill and stored in screw cap bottles at a temperature lower than -5 C. For the analysis of a segregating population compris- ing 834 clones from 18 families, 180 samples were randomly chosen. These potatoes were grown on the Montcalm Experi- mental Farm in 1970 on a Montcalm sandy loam with 800 lbs of 14-14-14 fertilizer at planting time, 130 lbs of N were added as a sidedressing and when spraying for insects and diseases. Irrigation consisted of an added 8 inches of water and after harvest the potatoes were stored at 40 F until November 1970. Preparation of samples: From each clone 5 tubers were chosen randomly. They were thoroughly washed, cut longi- tudinally in two parts and from one part 1 to 2 mm thick slices were removed with a potato chip slicer. The slices were put immediately on a layer of dry ice where they froze within 3 to 5 minutes. The samples, consisting of 35 slices of each clone, were kept in a freezer at -20 C until they could be freeze dried at a plate temperature below 100 F for 48 hours. After freeze drying and grinding in a Wiley mill through an 80 mesh sieve, the samples were stored in screw cap bottles at -5 C. 4 ,l-..|1 \lll‘ld af|lio till ’11:; RESULTS AND DISCUSSION Criteria chosen to judge the different procedures For uniform conditions the following statistical hierarchic assay structure was applied: Figure l: Hierarchic assay structure Varieties V1 . . . Samples Concentrations Replications Model: Yijkl = u + oi + Sij + Cijk + eijkl where: Yi'kl is the 1th observation in the kth concentra- 3 tion in the jth sample of the ith variety u is the parametric mean of the population “i is the fixed variety effect i' is the random contribution for the jth sample 3 of ith variety Ci'k is the random contribution for the kth con- ) centration in the jth sample of the ith variety ei'kl is the error term of the measurement of the J 1th test tube in the kth concentration in the jth sample of the ith variety 16 17 It is assumed that: Sij N N(O,o§) ‘ 2 Cijk N N(O,oc) e. N N(0,OZ) 1jk and oi are fixed i = l . . . . . v, v = 6 (varieties) j = l . s, s = 2 (samples per variety) k = l . . . . c, c = 3 (concentrations within one sample) 1 = l . . . r, r = 2 (replications within one con- centration level) The basic computations were conducted as described by Sokal and Rohlf (1969 a). During this study the time required for analyses was recorded. Table 2. Methionine analysis of 6 cultivars conducted with L. mesenteroides P-60, acid hydrolysis Source df MS FS EMS Among *** 2 2 2 2 varieties 5 2.0708 0.4142(1) 44.54 a + 20C + 608 + 12Kv Among 2 2 2 samples 6 0.0559 0.0093(2) l a + 20C + 605 Among con- * 2 2 centrations 24 0.2541 0.0106(3) 2.94 o + 20C "1th1n c°n' 36 0.1302 0.0030(4) oz centrations * as has a - 0.05 a 8 0.01 a - 0.001 a = Probability of error 18 From the components of the expected mean squares shown in Table 2 the single variance components can be derived: - MS (4)]/2 02 . MS (4) 6g . [MS (3) a; - [MS (2) - MS (3)1/6 K6 - [MS (1) - MS (2)1/12 in numbers: equation (1): equation equation (3): as equation or expressed as percentages of a variety .2 °y 32/scr A2 oC/sc .2 08/5 (2): CC (4): K 0.00362 0.00349 0.3347 0.000884 - 100 % variance 0.000302 0.000582 34.16% variance 65.84% variance 0 % variance Significant differences among varieties equation (1) equation (2) equation (3) equation (4) mean: of a variety mean within concentrations among concentrations among samples Tukey's multiple comparison procedure was chosen for this purpose: Least significant range: LSRa . Q(v ,k) x 57 19 v = n-l . 11, k = 6, 0a 3 0.01(11,6) - 6.247 59' JMS(2)7n - JUTUU937IZ - 0.028 LSR - 6.247 x 0.028 - 0.175 a- 0.01 Results: ug methionine per mg d.m. of 6 cultivars Merrimack M.S. Russet M.S. M.S. M.S. 58 711-3 Burbank 709 322-6 321-05 1.46 1.15 1.13 1.11 . 1.0 0.91 I a a = 0.01 = Probability of error Table 3: Recovery control of methionine Materials Added Recovered Recovery methionine methionine in % mg/g d.m. mg/g d.m. Potato starcht l 0.8 80% Merrimack .58 potato flow 0.5 0.48 96% * mean of two tests The reCovery of methionine added before hydrolysis was better in potato flour (Merrimack 58) than in pure potato starch. Optimal allocation of resources The assay structure was reorganized to determine the most efficient combination to give a variance less than or equal to a selected variance of the variety mean. 20 equation (6) 8;'- d2 3;'- estimated variance th of a variety mean A I equation (6) o; - 0.2972 = 0.01 t . 2.1 2 x 2 12 ‘ d = 0.297 - least sig- nificant difference between two varieties Computations of total time CO, 5', c', and r' Sokal and Rohlf (1969 b) give the equations as shown below. CO - cls' + czs'c' + c3s'c'r' equation (7) 8; - 82/5' + ai/s'c' + oZ/s'c'r' equation (8) t “2 “2 ‘2 I ‘2 I I ' s = l/o? [as + oC/c + o /c r ] equat1on (9) c' = «c SZ/c 32 I e uation (10) 1 c 2 s q r' = /c oz/c o2 e uation (ll) 2 3 c . 9 c1, c2 and c3 are defined on page 32 and their values are given in Table 9. 82, 83, and a; could be calculated from the analysis of variance tables. Finally, 5', c', and r' are the new numbers of samples per variety, concentrations within one sample, and replications within one concentration level, respectively. equation (10): c' = x . x = "big" c' + 2 equation (11): r' = x . x . = 0.58 r' + l equation (9): equation (7): s' B 1/0.01[0+0.00349/2+0.00362/2] - 0.35 C0 21 - 15 x 1 + l x 2 + 4 x 2 s'+1 25 min. In order to check the variance we take equation (8): A2. a. - 0/1 + 0.00349/2 + 0.00362/2 = 0.00174 Y 2 The new 1 sample (8') assay structure would comprise: A 0 6y - 0.00174, smaller than requested (0.01) 2 concentration levels per sample (c') 1 replication per concentration level (r') The time involved for analysis of one variety after optimal allocation would be 25 minutes, compared to 84 minutes using the basic structure as given on page 16. Methionine analysis of 6 cultivars conducted with S. zymogpnes, acidfihydrOIysis Assay structure, model and assumptions of the analysis of variance are the same as given on page 16. Table 4: Analysis of variance of methionine content estimated by means of S. zymogenes, acid hydrolysis Source df SS MS FS EMS eggggties 5 2.4130 .4826(1) 41.25"’ a2 + 263 + 60: + 12x3 Qflggfes 6 .0699 .0117(2) 1.26 oz + Zoé + 60% égggfiaggggs 24 .223 .0093(3) 3.1" 02 + Zaé Within C°“' 36 .1086 .003 (4) OZ centrations 22 From the components of the expected mean squares shown in Table 4 the single variance components can be derived analogically as in test 1: equation (1): 32 - 0.003 equation (2): = 0.0032 = 0.004 A2 00 . , A2 equat1on (3). OS 2 equation (4): KV = 0.0392 Expressed as percentages of a variety mean: equation (5): 32 - 0.00098 100 % variance of a variety Y me an 82/scr = 0.00025 25.5 % variance within concen- trations 554.08% variance among concen- aé/sc = 0.00053 trations 35/5 8 0.0002 20141% variance among samples Significant differences among_varieties Here again Tukey's multiple comparison procedure has been chosen. LSRa = Qa(v,k) x 5? Q6 - 0.01(11,6) = 6'247 s? - JMSIZS7n =JU.UII77I2 - 0.031 LSRa a 0.01 = 6.247 x 0.031 = 0.194 VBEEEEEEF pg methionine per mg d.m. 6 cultivars 23 Merrimack M.S. M S. Russet M.S. M S 53 711-3 709 Burbank 322-6 321-65 1.74 1.73 1.47 1.42 1.35 1.25 R *a - .01 Table 5: Recovery control of methionine Materials Added Recovered Recovery methionine methionine in % mg/g d.m. mg/g d.m. Potato starch 2 2.02 101 M.S. 321-65 flour 1 1.06 106 Table 5 shows a satisfactory recovery of methionine. Optimal allocation of resources The analogue calculations as on pages 20 and 21 were performed with the values obtained from this method: 3;, remains 0.01 3.5 , c' + 2 0 ll equation (10): equation (11): r' 0.484, r' + 1 equation (9) : s' 0.71 , 2' + 1 equation (7) : CO 25 minutes In order to recheck the variance take: A 7 equation (8) : a; - 0.0071, smaller than required (0.01) 24 The new assay structure, given a maximal variance of a mean A 0 (a; :_0.01), would comprise 1 sample (5') 2 concentration levels per sample (c') l replication per concentration level (r') and the time involved for the analyses of one variety after optimal allocation of resources would be 25 minutes compared to 84 minutes using the basic structure as given on page 16. Methionine analysis of 6 cultivars usipg_§. zymogpnes, and enzymatic digestion In contrast to the acid hydrolysates, the enzymatic digests were not filtered and were therefore turbid, even when diluted in the test tubes. An attempt was made to correct this basic turbidity. Some tests have shown that the basic turbidity of digested potato starch samples and digested potato samples is basically the same. As concentrations of potato starch digests increased, the relative absorbance became smaller. This was true for both, the inoculated and the uninoculated starch samples (Figure 2). To correct both, the basic turbidity and the decreased absorbance at higher concentration levels, the inoculated starch sample at a given concentration was subtracted from the same concentration of a sample with methionine ‘Ill '\ 'III‘IIv‘ 4.! I'lllfllllllllll.l I lll:|1l| IIIIII‘tnIl 25 Figure 2: Absorbance curve of uninoculated and inoculated potato starch samples partially supplemented with methionine inoculated supp me ted with 2 ug theoretical ' met/mg d.m. theoretical ./ I standard 1.1- ”’ ’ I I inoculated, no 91 upplemented O 7. ’1’ uninoculated , .54 ’ ot supple- mented Absorbance at 580 mu I; 10 20 30 40 pg suppl. * u *- % i : met per 1 2 3 4 test tube ml digests/ test tube supplemented potato starch (Table 6). This procedure also included the correction for methionine contained in the 4% papain enzyme solution. Table 6: 26 Procedure to correct basic turbidity Digests per Inoculated potato starch supplemented with 2 pg met/mg d.m. Inoculated potato starch test tube Absor- A - gross Absor- B - tare A-B - bance, met, bance met, net met m1 mg d.m- .580 mu. ug/mg d.m-.580.mu ug/mg d.m. ug/mg d.m. l S .77 .6 3.95 2.05 1.90 l S .79 .59 2 10 1.03 .75 3.68 1.78 1.90 2 10 1.025 .73 3 15 1.205 .84 3.42 1.5 1.92 3 15 1.2 .835 4 20 1.33 .93 3.22 1.47 1.75 4 20 1.36 .9 Recovery of methionine from the supplemented starch samples was 93.5%. In all tests where the "available" methionine was determined, two potato starch samples were prepared to correct the gross value of the potato samples. 27 Table 7: Analysis of variance of methionine content estimated with S. zymogenes, enzymatic digestion Source df SS MS PS EMS Am°08 . *4 2 z 2 2 var1et1es 5 3.381 0.6762(1) 15.333 0 + 20C + 608 + IZKV Among 2 2 2 samples 6 0.2644 0.0441(2) 1 o + 20C + 6oS Among con- *,* 2 2 centrations 24 1.4056 0.0586(3) 9.849 ' o + 20C Within con- 2 centrations 36 0.2142 0.0059S(4) o From the components of the expected mean squares shown in Table 7 the single variance components can be derived: 2 equation (1) = 8 = 0.00595 equation (2): 8E - 0.0263 equation (3): 3g - 0 equation (4): K3 - 0.0392 Expressed as percentages of a variety mean: equation (5): 8? - 0.004819 = 100 % variance of a variety y mean . 82/scr = 0.000496 - 10.51% variance within con- centrations ‘ Gg/sc = 0.004383 = 89.83% variance among con- centrations 33/5 = 0 = 0. % variance among samples 28 Significant differences Tukey's multiple comparison procedure has been applied: LSRa = Qu(o,k) x 5? Q6 = 0.01 (11,6) = 6°247 s? = JM31257n = J0.00441712 = 0.0606 LSRa 3 0.01 = 6.247 x 0.0606 = 0.379 Results: pg methionine per mg d.m. of 6 cultivars Merrimack M.S. M.S. Russet M.S. M.S. 58 711-3 322-6 Burbank 321-65 709 * 1.86 1.53 1.53 1.36 1.33 1.17 *u = .01 Tabl§_§; Recovery control of methionine Materials Added Recovered Recovery methionine methionine in % mg/g d.m. mg/g d.m. Potato starch 2 2.09 104 Merrimack 58 1 .93 93 flour Table 8 shows that the recovery of methionine was within reasonable limits. Optimal allocation of resources The analogue calculations as on pages 20 and 21 were performed with the values obtained from this method. 29 With given value for 8;'- 0.01 equation 00): c' "big" , c' + 3 equation.al): r' 0.24 , r' + 1 equation (9): 5' 1.075 , s' + 1 equation (7): CO 20 minutes In order to recheck the variance we take equation (8): 8%' 0.0175 4 0.01 The new assay structure would comprise: 1 sample (5') 3 concentration levels per sample (c') 1 replication per concentration level (r') and the time involved for the analysis of one variety after optimal allocation of resources would be 20 minutes compared to 64 minutes following the basic structure as given on page 20 and 21. mocofiu epmuom 30 m-HHn .m.2 mos .m.z o--m .m.z mo-H~m .m.2 mm .uhoz .m.m o a U m < _a u m < a u < a u < a m < u m < . v.0 r r m.o I II I l j. I F LL .Né f f r r: In [I J E .oé 2:: £33: eofioe E roé wouwvfixo .xsmmumoumsouso owcmaoxo :oH “ o mfimxflouwkn Owumezuno .mocoonxN .m u u u mwmxaopvxc kum .mocomosxn .m H m t < mwmxaouwxn meow .mowfiououcomoe .4 ” mwoguoe Ham mo ocflcoflzuoe mo memos Hm>fiufizu ”m oumwwm °m'p Bm/autuotqiem-I 3n 31 mocoao camped m-HHA .m.2 . ace .m.z o--m .m.z me-H~m .m.2 mm.eeoz .m.« o m4< m < m < m < m < m < we.o I... l L I a Tw.o T rL IL rL 1~.H L I .L . no.H t flfiaee .soeexv coneoe eoneoaxo .xgmmhmoumfiouzu omcmzuxo :oH ” r mfimxaouvxn wwom .movfiopopcomoe .4 u ocflumxo mo memos pm>fiwflsu ”v ohswfim 'm'p Bm/aurisKo-I 3n 32 mmeHopuxg oHumexuco was monomosxn .w meHmz mmeHmcm ochoHnuoE u m.H mmeHouvzn uHom paw mocmwoemu .m mnHm: mmeHwnw ochnguoa ” ~.e mmeHOHvxa vHoe was mopHououzomoE .H mnHm: «meHmcm ochoHsuoe " H.H ficoHumuHHaou H mo «coucou ocHeoHAHoE on» oueHsoHeo one .ooeenuomnm one use» .ouHHuoum .oupoan on common 05H» movaHoeHv ”no {it HHo>oH :oHumupcoucoo H now voquaon 05H» HenoHquvm movsHuaHU "No «a HoHaEmm H mo oaemxHOHvxs on» oummonn can :mHoz op wowoo: osHu movsHocHV "Ho fl 5H.cH Hm.m~ 0H.¢m omooo.o mmooo.o Nomooo.c a a a maoHueuHHnox «¢* «yak «Ci nw.mw wo.em ew.mo mmeoo.o mmooc.o. Nwmooc.o ««H “:H «cH meoHumaucoueoo o He.e~ o o Nooo.o o «m .mH .mH moflosem ooH coH ooH Nmeco.o mmooo.o ewwooo.o xuoHum> m.e ~.H . .H.H. m.H . ~.H , Hep .m.H., N-H , HeH w :H ousHomnm mousaHs :H zoos xulom> uHca Ho>oH Hon «HaouHuu m we mucoeogsou ooemHue> wouHscoH oaHH H when..wanmoH_vouHueaalw. um_oHne 33 Table 10: Summarized results, part II Criteria T.l T.2 T.3 Variance of a variety mean 0.028 0.031 0.0606 in potato starch 80 101 104 Recovery in % in whole potato 96 106 93 original structure 84 84 64 Total time needed for optimally allgca- 1 variety ted resources 25 25 20 in min. optimally allgga- ted resources 25 25 15 * . . . . .2 cond1t1on : var1ance of a variety mean a? < 0.01 equation (6) least significant difference d : /07088—= 0.297 ** A condition : variance of a variety mean a; :_0.02 equation (6) least significant difference d‘: V0.176 = 0.42 Discussion and conclusions of methods evaluating methionine Generally speaking all methionine determinations by S. zymogenes were higher than those by L. mesenteroides P-60 (Figure 3). Better recovery of supplemented methionine, which ranged from -3 to 24%, could be a partial explanation (Table 10). The overall means in T.l, T.2, and T.3 were 1.13, 1.49 and 1.47 ug methionine per mg d.m., respectively. A 24% increase of the mean value of T.1 would yield 1.40 ug methionine which is still beyond those of T.2 and T.3. 34 On the other hand, differences have been reported due to different bacteria species. However in T.1 and T.2 the results of the single varieties are prOportional. The results obtained with enzymatic digestion were lower but not exactly proportional as would be expected when the difference was considered in digestibility or biological availability of the individual amino acids from clone to clone. It was surprising to observe that the assay mean of T.3 (1.47 ug met/mg d.m.) was almost as high as in T.2 (1.49 ug met/mg d.m.) since the "available" methionine was expected to be lower than the "total" methionine. But what is the actual methionine content? This question arose when the methionine evaluated by microbiological procedures was compared to that evaluated by chemical methods (Kaldy, 1971). Here, with the exception of cultivars 322-6 and 321-65, all values were higher than those evaluated in T.3. This was to be expected since methionine was oxidized before the acid hydrolysis and all methionine should have been re- covered. In four cases the results estimated by chromatography and those by S. zymogenes and acid hydrolysis were within a range of 10%. In all cases L. mesenteroides gave the lowest results. The potato tuber contains some methionine sulfoxide in its tissue (Dent g1 31., 1947; Payne 21'g1., 1951). lil‘lllll'll fll ['1‘- ll I ll .1. All In. 11 1 ill. I|Iu| [III II- ‘Ir 35 Furthermore it has been reported (Block, 1956 a) that during acid hydrolysis some methionine would be oxidized to methionine sulfoxide. This oxidized methionine probably was included in the results evaluated by the ion chroma- tographic procedure of Kaldy (1971). Possibly, some bacteria can utilize methionine sulfoxide. The recovery from S. zymggenes for both, the supplemented potato starch and whole potato (Table 10) was better than from L. mesen- teroides. Therefore it was possible that S. zymogenes utilized methionine sulfoxide while L. mesenteroides did not. This hypothesis would explain the fact that the results were quite close when estimated by S. pymogenes '(acid hydrolysis) and ion exchange chromatography (Kaldy, 1971). From the point of view of economics S. zymogepes (enzymatic digestion) needs only 60% of the time required for L. mesenteroides and is more efficient (at a given 8% < 0.02). This is clearly shown in Table 10. Cystine analysis conducted by using L. mesenteroides P-60, acid hydrolysis Assay structure, model and assumptions for analysis of variance are the same as given on page 16. 36 Table 11: Analysis of variance of cystine estimated by using L. mesenteroides P-60, acid hydrolysis Source df SS MS FS EMS Among varieties 5 0.4526 0.0904 (1) 97.2*** o2 + 2oé + 60; + lZKé Among 2 2 2 samples 6 0.0056 0.00093(2) 1 .o + 20C + 6oS Among con- 2 2 centrations 24 0.0999 0.0042 (3) 1.5 o + 20C Within con- 2 centrations 36 0.1021 0.0028 (4) o From components of the expected mean squares shown in Table 11, one can derive the single variance components as in test 1. equation equation equation equation (1): 82 = (2): 85 = .62- (3). OS - . 2.. (4). KV - Expressed as percentages equation (5): 0.00035 0.00023 0.0007 0.0028 0.0007 0 0.0179 of a variety mean: 100 65.71% 34.29% variance variance tions variance tions variance of a variety mean within concentra- among concentra- among samples 37 Significant differences among varieties The multiple comparison procedure of Tukey has been chosen for this purpose. Least Signiflcant range, LSRa = Qa(v,k) x 57 k = 6 V = 11 Q0 = 0.01(11,6) ‘ 6:247 57 = /MSTTTTH = /07000937TZ = 0.0088 LSRa = 0.01 = 6.247 x 0.0088 . 0.05497 Resultsz' ug cystine per mg d.m. of 6 cultivars M.S. M.S. M.S. M.S. Russet Merrimack 322-6 711-3 321-65 709 Burbank 58 0.72 0.65 0.61 0.56 0.5 0.5 it *a = 0.01 Table 12: Recovery control of cystine Materials Added cystine Recovered cystine Recovery mg/g d.m. mg/g d.m. in % Potato starch 1 0.54 54 Merrimack 58 0.5 0.47 94 Discussion of method evaluating cystine It was very surprising to get such a large difference in recovered cystine between with cystine supplemented potato starch and with cystine supplemented 38 potato flour (Table 12). One reason for this phenomenon could be that during hydrolysis cystine was destroyed to a larger extent when it was together with pure potato starch than with all constituents of potato flour. Two-fold differences were observed between cystine contents evaluated by using L. mesenteroides and ion ex- change chromatography (Kaldy, 1970). Values reported in the literature range from 0.8 ug cystine/100 g protein (Schuhpan and Postel, 1957) to 1.6 pg cystine per 100 g protein (Hughes, 1958).1 Values obtained by Kaldy (1971) were in this indicated range. However cystine evaluated in this work were slightly lower than those reported by Schuhpan and Postel (1957) who in con- trast with Hughes (1958) and Kaldy (1971) did not oxidize the samples before hydrolysis. Cystine occurs naturally in the potato tuber (Barker and Mapson, 1952; Payne et a1., 1951). Since systine was oxidized to cysufine and finally to cysteic acid and deter- mined in this latter form, the naturally occurring cysteine in its oxidized state could also be found and therefore determined as cysteic acid. It has been reported (White 21 21., 1958) that bacteria used cysteine for the formation of methionine via homo- cystine. But since there was 20 times the necessary amount of methionine for normal growth in the cystine assay media, this reaction should not occur and therefore cysteine should have been used to form methionine. 39 On the other hand if methionine synthesis occurred from cysteine in the bacteria it would explain the higher recovery of supplemented methionine in the whole potato since cysteine was present in the whole potato but not in pure potato starch. However, this did not explain the large differences between the two methods of evaluating cystine. Optimal allocation of resources For cystine it would be sufficient to detect a differ- ence of about d = 0.14 ug cystine per mg d.m. between two varieties. equation (6) 8 = 0.142/2 x 4.4 = 0.00227 2! >7 d = 0.14, t 2.1 The analysis structure which should yield a variance of a variety mean of 8%.: 0.00227 would comprise: /15 x 0.00771 x 0 = "big" , c' + 2 equation (10): c' equation (11): r' = l , r' + 1 equation (9) : s' = 0.788 . , s' + 1 equation (7) : CO = 25 minutes In order to recheck the variance we use equation (8) : 3%, = 0.00175 2 0.00227 0.00175 < 0.00227 (requested variance 12' o— Y 40 Methionine analysis of 180 potato clones conducted py using S. zymoggnes enzymatic digestion Eighteen families with 10 clones each were tested for methionine. Assay structure: Families F1 . . . . . . . . . . . F18 Offsprings ’1””01 ”/,,92\\\‘ Samples 81 S2 S1 S2 Concen- \ / \ / \ / \ trations C1 C2 C1 C2 C1 C2 C1 C2 Replica- /\ \ /\ \ \ \ /\ \ tions R1 R2 R1 R2 R1 R2 R1 R2 R1 R2 R1 R2 R1 R2 R1 R2 a + O + Yijklm “" i ij Sijk * Cijkl + eijklm is the mth observation in the 1th concentration of the kth sample of the jth offspring in the ith family 0 is the parametric mean of the population a. is the ith family effect 01’ is the random contribution for the jth offspring of J the ith family Si’k is the random contribution for the kth sample in J the jth offspring of the ith family Cijkl is the random contribution for the 1th concentration in the kth sample of the jth offspring in the ith family 41 is the error term of the mth test tube's measurement of the 1th concentration in the kth sample of the jth offspring in the ith family eijklm It is assumed that: oi are fixed 2 Oij m N(O,o ) s m N(O oz) ijk ’ K 2 2 In this test: i = 1 . f, f = 18 (families) 3 = l o, o = 10 (Offsprings) k = 1 s, s = 2 (samples) 1 = 1 c, c = 2 (concentrations) m = 1 r, r = 2 (replications) This 5-1evel nested analysis of variance was computed from the analogue expansion of the 4-level nested analysis of variance described in Sokal and Rohlf (1969 a). All analyses were performed in five assays. Values received were corrected by means of a standard variety analyzed in each assay. The media used was prepared at one time and kept frozen until used. Samples whose concen- tration values differed more than 15% were repeated. The same is true for differences between the two samples of one variety. 42 No . Hmvwaoo.o ammo.m ONN .meoeoeeeeou -aoo canoe: woN + No mmme.oa HeVeHmo.o NmNm.oN oem maoaoeeeaoo «ea unou wdos< woe + wom + o memo.H Hmveewo.o Hmefi.mH owe moHeEem N N mdofi< cow 4 woe + ooN + o emmm.- HNvmmmm.H emom.mHm NeH mmeaaom N N N N «a. -ewo maoa< ex on + one + woe . uoN + o eomN.N Hevmeom.e Hmem.ee AH moHHHEem N N N N N «a wcoa< mZm mm m: mm awe ouusom :oHumomHe uHumeznco .mocmwosxm,.m maHms kn gone mmcHummmmo OH :uH3 moHHHEmm wH mo mucoucou ochoHnuoE mo oocwHum> mo mmequ< umH oHan 43 The results were calculated as described in test 3. From the components of the expected mean squares shown in Table 13 the single variance components can be derived: 82 = MS (5) = 0.0078 8% = [MS (4) - MS (5)1/2 = 0.0368 8% = [M8 (3) - MS (4)1/4 = 0.0008 83 = [MS (2) - MS (3)]/8 = 0.2319 K% = [MS (1) - MS (2)]/80= 0.0300 or expressed as percentages of the whole assay mean: 8 = GZ/foscr + Gé/fosc + Gé/fos + ‘ 3.3 y = population mean 1.2 1.5 1.8 ug l-met/mg d.m. Frequency Frequency Frequency Frequency Frequency Figung_§: r—aNuA ~1th b—‘NM-D- I-‘Nm-A HIQIA-b _ 1,1 1 clones according to families #850 1.2 1.8 2.4 3.0 ug l-met/mg d.m. l l l j #852 L 1.2 1.8 2.4 3.0 ug l-met/mg d.m. #854 F. ‘ ['1 Fl r r. 1.2 138'234 3.0 ug l-met/mg d.m. #856 ‘ l f”! U I I '172'.1.8 2.4 3.0 pg l-met/mg d.m. #858 F“ l 1.2 1.8 2.4 3.'0‘ r pg l-met/mg d.m. Frequency Frequency Frequency Frequency Frequency Frequency distributions of methionine of 180 —: #851 4: 3i 2* ‘1—1 1. IF! TI I IT 1.2 1.8 2.4 3.0 ug 1-met/mg d.m. #853 4. 3‘ 2‘ 1‘ 1.2 1.8 2.4 3.0 ug l-met/mg d.m. #855 44 F1 3- 2. 1‘ f '1.2 138'2'.4'3'.011 ug l-met/mg d.m. #857 4. 3‘1 24 1‘1 I I 1.2 1.8 2.4 3.0 ug l-met/mg d.m. #859 fi 41 3d 2- 1‘ J—l U U T 1.2 138'2.4 3.'0' ‘ ug l-met/mg d.m. ‘l'lr 47 Figure 6: Cont. #860 #862 >~ 84: 24‘ - C134 123-4 5’24 324 8‘ J H 1_. . '1VYTT II—' wjjIj‘ '1.'2 1.8 2.4 3.0"T 1.2 1.8 2.4 3.0 pg l-met/mg d.m. pg l-met/mg d.m. 5‘ .1 #863 5‘4. #864 c 4 c 83) 83" 8‘2 1 3‘2‘7 81‘ al‘ .l-III IIIIIrIrII 1.2 1.8 2.4 3.0 1.2 1.8 2.4 3.0 pg l-met/mg d.m. pg l-met/mg d.m. #866 #867 §3~ 33% 0'2 7 8‘2 7 El“ 521‘ 'Ij II III—r'r ‘ 1.2 1.8 2.4 3.0 1.2 1.8 2.4 3.0 pg l-met/mg d.m. pg l-met/mg d.m. 54. #868 5-4. #870 531 53-1 3‘21 [ ’* §21 1 I 81“ I'll El‘fi—LLLLH-FT-P LL. 11"I'Ur'1 1.2 1.8 2.4 3.0 1.2 1.8 2.4 3.0 pg l-met/mg d.m. pg l-met/mg d.m. 48 of each family. For example in #857 there is little segregation which is contrary to #851, #854, #864, #866 and #867 which have clones with methionine contents ranging from 1.2 up to 3.6 pg l-met per mg d.m. REFERENCES Albanese, A. A., and Orto, L. A. 1968. Proteins and amino acids. In "Modern Nutrition in Health and Disease," ed. M. G. Wohl and R. S. Goodhart, p. 107. Lea and Febiger. Barker, J..and Mapson, L. W. 1952. The absorbic acid content of potato tubers. III The influence of storage in nitrogen, air, and pure oxygen. New Phytologist 51, 90-115. Borgstrom, G. 1969. Too Many: A Study of Earth's Bio- logical Limitations, pp. 40-45. Macmillan Ltd., London. ‘ Barton-Wright, E. C. 1952. The Microbiological Assay of the Vit B-Complex and Amino Acids. Pitman, London. Block, R. J. 1956 a. Amino Acid Handbook, pp. 131, 132. Charles C. Thomas, Publisher, Springfield, Illinois, USA. . 1956 b. Amino Acid Handbook, p. 51. Charles C. Thomas, Publisher, Springfield, Illinois, USA. Boyne, A. W., Price, S. A., Rosen, C. D., and Stott, J. A. 1967. -Protein quality of feeding-stuffs, 4., Progress report on collaborative studies on the microbiological assay of available amino acids. Br. J. Nutr., 21, 181-206. Dent, C., Stepka, W., and Steward, F. 1947. Detection of the free amino acids of plant cells by partition chromatography. Nature, 160, 682-683. Difco Manual of Dehydrated Culture Media and Reagents for Microbiological and Clinical Laboratory Procedures. Ninth Edition, 1969, p. 232. Difco Laboratories, Inc., Detroit, Mich. Evans, R., and Butts, H. A. 1948. Studies on the heat inactivation of lysine in soy bean oil meal. J. biol. Chem. 175, 15-20. 49 50 Evans, R., Bandemer, S. L., and Bauer, D. H. 1962. Effect of heating soybean proteins in the autoclave on the liberation of cystine and methionine by several digestion procedures. Agricultural and Food Chemistry, 10, 416. Evans, R. J., and Bauer, D. H. 1970. Working paper. Michigan State University, Department of Biochemistry, E. Lansing, Mich. Food and Agriculture Organisation of the United Nations. 1957. Protein requirements. FAO Nutritional Studies No. 16, Rome. Ford, J. E. 1960. A microbiological method for assessing the nutritional value of proteins. Br. J. Nutr. 14, 485-497. . 1962. A microbiological method for assessing the nutritional value of proteins, 2., The measurement of "available" methionine, leucine, isoleucine, arginine, histidine, tryptophan, and valine. Br. J. Nutr. 16, 409-25. Guggenheim, K. 1970. Evaluation of Novel Protein Products. Proceedings of the International Biological Program (IBP) and Wenner-Gren Center Symposium held in Stock- holm, Sept. 1968, p. 238. Edited by A. E. Bender, B. Loefqvist, R. Kihlberg and L. Munck. Pergamon Press, Oxford, N.Y., Toronto, Sydney, Braunschweig. Hughes, B. P. 1958. The amino acid composition of potato protein and of cooked potato. British J. Nutr. 12, 188-195. Kaldy, M. S. 1971. Evaluation of the potato protein by amino acid analysis and dye-binding. Ph.D. Thesis, Michigan State University, E. Lansing, Mich. Kidder, G. W., and Dewey, V. C. 1949. Studies on the bio- chemistry of Tetrahymena. XIII. B vitamin requirements. Arch. Biochem. 21, 66-73. Lewis, 0. A. M. 1966. Short ion-exchange column method for the estimation of cystine and methionine. Nature, 209, 5039-5040. Lyman, C. M., Moseley, 0., Wood, 8., and Hale, F. 1946. Note on the use of hydrogen peroxide treated peptone in media for the microbiological determination of amino acids. Arch. Biochem. 10, 427-42. 51 Mulder, E. S., and Bakema, K. 1956. Effect of the nitrogen, 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, VII, No. 2, 135-166. Payne, M. G., Fults, J. L., and Hay, R. J. 1951. Free amino-acids in potato tubers altered by 2, 4-D treat- ment of plants. Science (Washington) 114, 204-205. Rios, B. J. 1969. Protein value of potato and navy bean powders: Nutritional evaluation using the meadow vole (Microtus pennsylvanicus), p. 49. Master Thesis, Michigan State University, E. Lansing, Mich. Rosen, G. D., Stott, J. A., and Smith, H. 1960. Proc. int. Congr. Nutr. V. Washington, p. 72. ; Schram, E., Moore, 8., and Bigwood, B. J. 1954. Chromato- graphy determination of cystine and cysteic acid. Biochem. J. 57, 33-37. Schuhpan, W., and Postel, W. 1957. Ueber die biologische Wertigkeit des Kartoffeleiweisses. Die statistische Streuung der Anteile der exogenen Aminosaeuren am Rohprotein in Abhaengigkeit von genetischen und oekologischen Faktoren Naturwiss. 44, 40-41. Schuhpan, W. 1958. Proteins et amino-acids. Teneurs en amino-acids indispensables des vegetaux alimentaires et de leur diverses organes. Qual. Plant. Mater. Veg. 3-4, 19-33. Sokal, R. R., and Rohlf, F. J. 1969 a. Biometry, pp. 256- 287. W. H. Freeman and Company, San Francisco. 1969 b. Biometry, pp. 287- 295. W. H. Freeman andflCompany, San Francisco. Spackman, D. H., Skin, W. H., and Moore, 8. 1956. Auto- matic recording apparatus for use in chromatography of amino acids. Federation Proceedings, 15, 358. Spies, J. R., and Chambers, J. R. 1948. Chemical deter- mination of tryptophan. Anal. Chem. 20, 30-39. Spies, R. J. 1967. Determination of tryptophan in protein. Analytical Chemistry, Vol. 39, Nr. 12, 1412-1416. Steel, B. F., Sauberlich, H. E., Reynolds, M. S., and Baumann, C. A. 1949. Media for Leuconostoc mesen- teroides P-60 and Leuconostoc citrovorum 8081. J. Biol. Chem. 177, 534-544. 52 White, A., Handler P., and Smith, E. L. 1968. Principles of Biochemistry, p. 566. McGraw-Hill Book Company, N.Y., Sydney, Toronto, London. Williams, H. H. 1959. Amino acid requirements. J. Americ. Dietet. Ass. 35, 929-933. Watts, H. J., Booker, L. K., McAfee, J. W., Graham, D. C. W., and Jones, Jr. F. 1959. Biological availability of essential amino acids to human subjects. 11. Whole egg, milk, and cottage cheese. J. Nutr. 67, 497-508. APPENDICES 54 Appendix 1: Basal medium for S. zympgenes (1) Glucose (g) KZHPO4 (g) Citric acid (g) Sodium acetate (trihydrate) (g) Tween 80 (ml)* Solution of mineral salts (m1)H Adenine (mg) Guanine (mg) Uracil (mg) Xanthine (mg) Thiamine (mg) Pyridoxal ethylacetal hydrochloride (mg) Riboflavine (mg) Nicotin acid (mg) Calcium pantothenate (mg) p-Aminobenzoic acid (mg) Folic acid (mg) Biotin (pg) Ascorbic acid (g) Vitamin B12 (pg) pH adjusted to 7.2 with acetic acid Water added to 200 ml 8 0.5 g; ZnSO 0.25g; CuSO to cl (1) Boyne 31 a1. (1967). * Polyoxyethylene sorbitan mono-oleate * Contained MgClZ .6H20, 20g; CaClz, 5g; FeCl3 4 .7H20, 0.5g; MnSO4 .4H20, 0.25g; CoCl 4 .SHZO, 0.25g; V0804, 0.25g; Na2 ear. H N |-| «1 N o o o ONNNNNNU‘IU‘IU‘IU’IOH O p... O 2 NO U1 .6H2 .6H 0. 2 M004, 0.25g; dissolved in l l distilled water with addition of N-H7SO 0. 4 55 Appendix 2: Amino acid supplement for §1_zymogenes (l) l-Glutamic acid (g) l-Leucine (g) l-Isoleucine* (g) 1-Va1ine (g) 1-Lysine hydrochloride (g) NNNNNNNNNNMMMU‘IMM l-Alanine (g) l-Aspartic acid (g) 1-Arginine hydrochloride (g) Glycine (g) l-Cystine (g)** l-Serine (g) l-Tyrosine (g) l-Proline (g) l-Hystidine hydrochloride (g) l-Phenylalanine (g) l-Threonine (g) OOOOOOOOOOOOOOOOH O. .. ...... ... l-Tryptophan (g) pH adjusted with N-KOH to 7.2 Water added to 250 ml (l) Boyne 31.31. (1967). * Allo free an First dissolved separately in 10 m1 boiling water by addition of HCI. 56 Appendix 3: Citrate cyanide buffer Trisodium citrate (g) 5 Sodium cyanide (mg) 30 Dissolve in distilled water, adjust pH to 7.2 with N-H3P04 and dilute to 1000 ml. 57 Appendix 4: Basal medium and amino acid supplement for L. mesenteroides,met assay (2) mg dl-a-Alanine 200 l-Arginine HCl 242 l-Asparagine 400 l-Aspartic Acid 100 l-Cysteine 50 l-Cystine l-Glutamic acid 300 Glycine 100 l-Histidine HCl 62 dl-Isoleucine 250 dl-Leucine 250 l-Lysine HCl 250 *dl-Methionine 100 dl-Phenylalanine 100 l-Proline 100 dl-Serine 50 dl-Threonine 200 dl-Tryptophan 40 l-Tyrosine 100 dl-Valine 250 8 Total weight 3.1 Glucose 25 Sodium acetate 20 Ammonium chloride 3 m8 KH2P04 600 KZHPO4 600 MgSO4 7H20 200 FeSO4 . 7H20 10 MnSO4 4H20 20 NaCl 10 Adenine sulfate . H20 10 Guanine . HCl . ZHZO 10 Uracil 10 Xanthine 10 Thiamine . HCl 0,5 Pyridoxine . HCl 1.0 Pyridoxamine . HCl 0 3 58 Ca dl-Pantothenate Riboflavin Nicotinic acid p-Aminobenzoic acid Biotin Folic acid Pyridoxal . HCl Distilled H20 to (2) Steele e£_a1, (1949). i4 Omitted for 1-met assay OOOOHOO O O O 0 . LNOOHOU'HJI ml 500 59 .cqu .mnHmcm4 .m .qumHo>H:D oumum :mmHgon .xuumHEosuon mo ucosuhmmoo .uosem .: .4 mmHz paw mcm>m .O .m .ha mo Hmouhsou ecu gmsounu vo>Houom HmO mossy umou mo Honssz HE OOmH OOOH mum Omn mNO OOm op ousHHO O O mN.m m.e mn.m m OmN :H HEH eHo< UHHom m N mN.H m.H mN.H H mH m< eHum uHouconocHEm-O mH OH mn.m m.n mN.O m mH m< mw< munm Om ON m.NH mH m.NH OH uo\mn m.O :Hpon o0 oe mm on mN ON uu\w: OOH wHu< umcmuccmz OO OO mm om mN ON 00\w: OOH :H>meonHm ONH ON ON OO Om Oe uu\m: Om oumconuoucmm-mu Om ON m.NH mH m.NH OH uu\mn OON oconeHuxm mH OH mN.O m.n mN.o m UU\m: OON ocHEmth . . HHumHD om ON m NH mH m NH OH UU\mEH .ocHemso .ochoe< ON ON m.NH mH m.NH OH UU\mEOH ocH:0choE-He ONH ON ON OO Om OO uo\wsm.N oeHmone-H ON ON m.NH OH m.NH OH 8353 eaeoooaaae-HO HEOme HEOOm Hem.NON HEmNN Ham.NOH HEOmH eoumoeu NON:-o:oumoO OH NH m.OH O m.N O HO-O:z ON ON HN OH mH NH u<-mz mOO MOO mmm NON OON mON omoooHO OOm OON .mNH OmH mNH OOH meeoHeoaNeH HmcoHusHOm Muoum vohmmohm Eoum a: oemsu Hmv OO-O moeHoHoucomoE .4 How EsHeoE Hemmm ocHumxu esp mo :OHuHmomEou ”m xHecomm< 60 As indicated the amino acid supplement was partially replaced by hydrogen peroxide treated peptone prepared as described by Lyman £1 31. (1946): Preparation of hydrogen peroxide-treated peptone: Fifty g of bacto-peptone in 500 m1 of N-HCl were treated with 0.05 mol of hydrogen peroxide (5.7 g of 30% H202) and allowed to stand overnight at room temperature. The solution was then heated in a steam sterilizer at atmospheric pressure (100 C) for 30 minutes, cooled, neutralized with sodium hydroxide and steamed again, this time for one hour. The purpose of the second steaming was to decompose any hydrogen peroxide not used up by the oxidative reactions. The preparation was ready for use after diluting to a final volume of one liter. Preparation of the stock solutions (Evans and Bauer, 1970) dl-Methionine: 2.5 g dl-methionine was dissolved in 200 ml distilled water and warmed up on the steam bath. Before the solution was made up to a volume of 250 m1, 2 drops of conc. HCl were added. dl-Tryptophan: 2.5 g dl-tryptophan was dissolved in 150 m1 water with the aid of 5 drops conc. HCl and heated on the steam bath until completely dissolved. Then the solution was cooled to room temperature and made up to a volume of 250 ml. 61 l-Tyrosinez. 1.25 g l-tyrosine was dissolved in 400 m1 distilled H 0, a few drops of 12% NaOH added and made up to 2 a final volume of 500 ml. GlucoseJ sodium acetatel_and ammonium chloride were weighed out as required. Adenine,vguanine, uracil: 0.2 g of each was transferred into a 200 ml volumetric flask. The solution was warmed up on a steam bath and conc. HCl was added until the solution turned clear. Nicotinic acid: A separate stock solution was prepared by dissolving 0.5 g niotic acid in 500 m1 of distilled water. The stock solution was diluted one in ten. P-aminobenzoic acid: 50 mg was dissolved in 500 ml distilled water. Calcium-pantothenate: 50 mg was dissolved in 999 m1 dis- tilled water and 1 m1 of salt solution A was added. Pyridoxine: 50 mg was dissolved in 250 ml distilled water. Riboflavine: 1 ml of glacial acetic acid was added to 50 mg anhydrous riboflavine and heated over the steam bath in 499 ml water. Folic acid: 10 mg folic acid was dissolved in 10 m1 dis- tilled water containing a few dr0ps 12% NaOH. The stock solution was diluted 1 in 250. 62 Biotip; 25 mg biotin was dissolved in 50 ml 80% ethanol. The stock solution was diluted 1 in 100. Ihiaming: 50 mg was dissolved in 250 ml distilled water. Salt A: 25 g of each, KZHPO4 and KH2P04 were dissolved in 250 ml distilled water containing a few drops of conc. HCl and conc. H2804. Salt B: 10 mg MgSO4 . 7H20, 0.5g NaCl, 0.5g MnSO4 . ZHZO, and 0.5g FeSO4 . 7H20 were dissolved in 250 ml distilled water containing a few drops conc. HCl. All solutions described above were stored in the refrigerator.