In the following paragraphs a few examples will be given of the supplementation of foods and feeds with their first limiting amino acid.
A. CORN MEAL
Corn meal is an important foodstuff especially in the southern part of the United States. The first limiting amino acid of corn is lysine according to calculation based on its amino acid analysis as well as according to experimental results. Mitchell and Smuts (1932) obtained definite im-provement in growth when 0.25% lysine was added to an all corn diet containing 8% protein. Other investigators have carried out similar ex-periments but apparently graded levels of lysine were never fed in order to determine the extent to which an all corn diet might be improved by supplementation with the first limiting amino acid. Emphasis, instead, was placed on the simultaneous addition of several amino acids (Sure, 1953; Sauberlich et al., 1953) in order to provide all amino acids in at least the required amounts and in order to obtain growth equivalent to that obtained from a normal diet.
For the purpose of illustration, degerminated corn meal was chosen rather than whole corn. It was fed as 90% of a diet which contained all the essential vitamins, minerals, and 5% of fat. From calculation, based on its amino acid analysis (Edwards and Allen, 1958), about 0.03%
lysine would be needed to bring this amino acid in balance with trypto-phan, the second limiting amino acid. The design and the results of two rat growth experiments are seen in Table II. There is considerable uni-formity in these data which suggest that about 0.05% L-lysine · HC1, equivalent to 0.04% lysine, brought about maximum response. Weight gain in these experiments was improved 37% and 41%, respectively, due to balancing the protein with lysine. Supplementation of this diet with larger amounts of lysine depressed growth and feed efficiency. As the
406 HANS R. ROSENBERG
corn meal contained very little protein, 6.75% (N X 6.25), the animals responded readily to an induced amino acid imbalance (Rosenberg, 1959).
TABLE II
SUPPLEMENTATION OF CORN MEAL WITH L-LYSINE · HCla
L-Lysine-HC1
a Five-week data. Six male rats per treatment.
B. RICE
This example is taken from a publication of Rosenberg and Culik (1957). Earlier workers had not been able to demonstrate a beneficial effect from the addition of the first limiting amino acid (lysine) alone since they added to the low (7-8%) protein diet, the full requirement of lysine, 1.0% of the diet, as suggested by Rose (1937) for the growing rat. Thus an imbalance was created resulting in no improvement in the growth rate of the animals. Pecora and Hundley (1951), however, had made the important discovery that the combination of lysine plus thre-onine improved growth considerably in short term rat experiments. Ac-cording to amino acid calculations, however, lysine should be the first limiting amino acid in rice (Mitchell and Block, 1946).
In this example precooked rice was used rather than raw rice in order to test this food in the approximate condition in which it is consumed.
The design of the experiment in which the rice was fed as 90% of the diet and the responses at 5 weeks are shown in Table III. Highly
sig-TABLE III
SUPPLEMENTATION OF PRECOOKED RICE WITH LYSINE; EFFECT ON FIVE WEEKS' RAT GROWTH0
a Twenty animals per treatment.
AMINO ACID SUPPLEMENTATION OF FOODS AND FEEDS 4 0 7
nificant responses to L-lysine-HCl supplementation were obtained, best gains and efficiency of food utilization being observed at a level of 0.10%
of supplementary L-lysine-HCl for both males and females. Over-all growth is roughly twice as good on the optimally supplemented ration as on the adequately supplemented corn meal ration, yet it was only about half as good as that obtained with the fully supplemented bread diet or with the stock diet.
In order to carry the evaluation program beyond the initial stage of the short range rat growth test, a larger number of animals was studied over a half-year period. The growth curves for the group of animals on the basal rice diet and on the diet supplemented with 0.1% L-lysine-HCl
T 1 1 1 1 1 1 1 1 1 r
WEEKS ON EXPERIMENT
FIG. 8. Growth response of rats on precooked rice diet to supplementation with optimum level of L-lysine-HCl.
are reproduced in Fig. 8. The important observation, here, is that the animals consuming the properly supplemented rice diet have grown to essentially the same weight as similar animals do on a stock diet. On the other hand, the animals on the unsupplemented diet were distinctly ab-normal in size as well as in weight and appearance, with rough coats and scaly tails.
C. BREAD
This example is of particular interest because supplementation of various breads has been practiced commercially since 1954. This develop-ment is based on extensive studies over a ten-year period on the im-provement in the nutritional quality of bread by supplementation with lysine, the first limiting amino acid. It was realized from the start that because of differences in digestibility experimental studies should be
408 HANS R. ROSENBERG
carried out with bread, not with flour. It was also known from the work of earlier authors that lysine is somewhat subject to destruction by heat.
Initially, therefore, a study was undertaken to learn the extent of the destruction of the native lysine in the flour during the baking procedure (Rosenberg and Rohdenburg, 1951). Next, these authors (Rosenberg and Rohdenburg, 1952) determined the amount of lysine-HCl which, when added to commercial white bread, would give optimum response in the 5-week rat growth test. Table IV shows the results of a similar
TABLE IV
experiment run almost ten years after the first experiment with animals from the same colony and with a commercial white bread containing 5%
non-fat milk solids (Rosenberg, 1959). Addition of 0.4% L-lysineHCI to the 90% air dried bread diet gave maximum response for the males while 0.3% L-lysine-HCl was needed by the females. It is to be noted that at these levels of supplementation the animals grew as well as on the stock diet. It seems justified, therefore, to repeat the earlier conclu-sion: "These results suggest that, as far as rat growth is concerned, the only important amino acid deficiency in commercial bread is lysine."
These investigators proceeded then to a similar growth study ex-tended over 6 months, the results of which are seen in Fig. 9. Another group of animals, not shown in this graph, was fed the stock diet and grew as well as the animals on the bread diet fortified with the largest amount of lysine. Of particular significance are, perhaps, the results obtained from the addition of 0.2% lysine. The animals (mixed sex) grew during the period of early growth at an average rate of about 75%
iof that of a similar group of animals on the stock diet. This disadvantage
AMINO ACID SUPPLEMENTATION OF FOODS AND FEEDS 4 0 9
was essentially overcome by the time the animals were 6 months old.
Animals receiving less than 0.2% lysine supplementation, after 25 weeks' growth, did not attain weights comparable to those receiving the stock diet. It is also obvious that this relatively small addition, 0.2% lysine, to the basal diet was utilized very effectively. The protein efficiency was much improved over that observed with diets of lower lysine content and not greatly inferior to that found with larger lysine additions.
Breads were then baked from flour to which 0.25 pounds of L-lysine
•HC1 had been added per 100 pounds. The growth responses were found similar to those from bread to which the lysine had been added
0 5 10 IS 20 25 TIME IN WEEKS
FIG. 9. Twenty-five weeks' growth response of rats to bread diets containing increasing amounts of supplementary lysine.
after baking. On the basis of many experiments of this kind it seemed appropriate to consider 0.25% L-lysine HC1 as a reasonable addition to flour. The lysine content of the supplemented flour and bread compares favorably with accepted standards (Fig. 10). The flour thus supple-mented may also be considered to have been reconstituted to that of whole wheat as far as its amino acid pattern is concerned.
The results of a reproduction and lactation study with rats have recently been published (Culik and Rosenberg, 1958) in which the effects of commercial white bread containing 6% milk solids were com-pared with a similar bread baked from flour supplemented with 0.25%
lysine HC1 and with the stock diet. The comparison was carried out with groups of animals maintained on their respective diets from wean-ing until death and bred until fertility ceased. The first litter of the parent generation on each diet was raised to maturity and then mated.
(9.25) 7.0 6.0 h 5.0 h 4.0 h 3.0 h 2.0 h 1.0h
(WT. OF THREONINE PER GM . OF NITROGEN TAKEN AS 3.0 IN EACH CASE) jgggj WHITE WHEAT FLOUR WHOLE E6G EFFECT OF A DOING I CM. L-LYSINE · H Ct PER 4Θ CMS. WHEAT PROTEIN
H o 7.0 6.0 5.0 4.0 H3.0 -U.0 Hl.O ISOLEUCINE PHENYL- ALANINE + TYROSINE
METHIONINE THREONINE + CYSTINE TRYPTOPHAN FIG. 10. Proportions by weight of essential amino acids in white flour, egg, and meat protein compared with FAO reference pattern (based on data from Home Econ. Research Rept. No. 4, "Amino Acid Content of Foods/' U.S. Department of Agriculture, 1957).
2 C/3 o H «
AMINO ACID SUPPLEMENTATION OF FOODS AND FEEDS 4 1 1
From many observations over a three-year period, on the parent genera-tion as well as on their seven litters and on six successive generagenera-tions, involving ultimately several thousand animals, it was obvious that com-mercial bread as the only source of dietary protein did not support ade-quate reproduction. There was a reduced rate of conception as well as poor lactation, resulting in very low body weights.
Lysine supplementation of the bread improved reproduction and lactation performance considerably and uniformly. Figure 11 shows the average weights of the litters of the parent generation at 9 weeks of age.
In order to obtain continued reproduction, the females on the commercial bread diet had to be supplied with males from the stock colony for about
200
<
100 I
1
LITTER 4 5LILU
■ STOCK DIET
D LYSINE-SUPPLEMENTED BREAD DIET B COMMERCIAL BREAD DIET
FIG. 11. Growth response of seven litters of rats on commercial bread diets.
40% of their breeding since the males on the commercial bread diet had died early. Figure 12 shows a similar picture for the six successive gen-erations. During this work several symptoms of lysine deficiency were observed including nervousness, irritability, and perverted appetite. The rats chewed on everything they could reach and ate their own hair, thus denuding those parts of their body which they could reach. The hair accumulated in their stomachs and, upon death, hair boluses were found in all animals maintained on the commercial bread diet. No such hair boluses nor any of the other symptoms were seen in the animals reared on lysine-supplemented bread.
After these reports were published many laboratories here and abroad worked on this problem. The initial findings were confirmed in this country (Jahnke and Schuck, 1957; Westerman and his co-workers, 1957; Sabiston and Kennedy, 1957) and abroad (Hutchison et al., 1956)
412 HANS R. ROSENBERG
and extended. For example, it was shown that regardless of the level of non-fat milk solids chosen as addition to commercial bread, lysine is still the first limiting amino acid in bread and the nutritive value of bread can be improved substantially by lysine supplementation.
2001-100 h
1
1 H_
D
2 3 4 5 6 GENERATION
STOCK DIET
LYSINE-SUPPLEMENTED BREAD DIET COMMERCIAL BREAD DIET
FIG. 12.
bread diets. Growth response of six successive generations of rats on commercial D. ANIMAL FEEDS BASED ON CORN AND SOYBEAN OIL MEAL
1. Broiler Diets
To illustrate the use of methionine and of its hydroxy analog, two examples are given. The diets are a 24% protein commercial type 1958 broiler ration and the 21% protein Arkansas performance test diet (Stephenson, 1956), the compositions of which are shown in Table V.
The results obtained with these diets are seen in Tables VI and VII.
Groups of 70 day-old crossbred chicks (Vantress males X Nichols #12 females) were raised in floor pens for 9 weeks. With diets of this type 3-pound broilers are raised commercially in 8 weeks with a consumption of a little over 6 pounds of feed per bird. This would not be possible without the methionine supplementation which improves both growth and efficiency of feed utilization and is highly economical.
2. Turkey Diets
This example has been chosen to illustrate the saving in feedstuffs which can be accomplished by amino acid supplementation (Baldini et al., 1954). Turkey starter rations are usually formulated to contain 28-30% protein. Commercially, methionine is now added generally to turkey rations to improve the efficiency. The example in Table VIII
TABLE V COMPOSITION OF ANIMAL FEEDS BASED ON CORN AND SOYBEAN OIL MEAL Components Ground yellow corn Soybean oil meal, solv., 45% protein Soybean oil meal, dehulled 50% protein Alfalfa meal Fish meal Fish solubles Meat scraps Fat Limestone Calcium carbonate Vitamin supplement Dicalcium phosphate Salt, iodized Trace mineral mix Dried whey Butyl fermentation sol. Dried distillers' solubles
Com- mercial 1958 broiler ration0 50.67 —
26.50 3.00 5.00 — 3.00 5.00 — — X 1.50 0.30 — 3.00 —
2.00
Arkansas perform- ance test diet0 55\5 —
25.3 2.0 4.0 2.0 — 3.0 1.4 — X 2.0 0.5 2.0 2.0
Turkey starter 20% protein 60.57 32.00 —
2.50 — — — —
2.50 — X 1.25 0.45 — — —
Turkey starter 28% protein 37.57 55.00 —
2.50 — — — —
2.50
X 1.25 0.45 —
Pig starter 90.6 5.7 — — — — — — — 1.2 X 0.9 0.5 0.1 — —
>
§
o >g
C/52
g as o 3 o *l o o Ö C/3 > 2 o3
W8
a The commercial formulation of this diet contains 1-2 pounds of DL-methionine per ton of feed. & The Arkansas performance test diet contains an additional 2 pounds of DL-methionine per ton of feed.414 HANS R. ROSENBERG
shows that a 20% turkey starter ration supplemented with methionine and lysine may give as good a performance as a 28% protein diet sup-plemented with methionine. The latter combination was not improved by additional supplementation with lysine. Jersey Buff poults were fed
TABLE VI
SUPPLEMENTATION OF BROILER RATION WITH DL-METHIONINE
DL-Methionine
a When this diet is mixed commercially DL-methionine is added at the rate of 1-2 pounds per ton.
TABLE VII
THE METHIONINE RESPONSE IN THE ARKANSAS PERFORMANCE TEST D I E T
Weight gain
Supplementation of broilers at 9 e e Index of
to test diet weeks (gm.) Gain performance
~ 1582 SÜ51 63(3 0.05% DL-Methionine 1658 2.39 694 0.05% "Hydan"<* 1640 2.38 692
α "Hydan" — Du Pont's calcium methionine hydroxy analog.
TABLE VIII
SUPPLEMENTATION OF TURKEY STARTER D I E T WITH AMINO ACIDS
Average
20% Protein basal + 0.30% L-lysine-HCl 20% Protein basal -f 0.20% DL-methionine 20% Protein basal -f 0.20% DL-methionine
+ 0.30% lysine HC1 28% Protein basal
28% Protein basal -f 0.20% DL-methionine
the diets shown in Table V. The saving in feed possible by reduction of protein level to 20-22% and supplementation with both lysine and methionine has been confirmed (Fisher et al, 1956). There are great opportunities for further improvements in turkey diets by increasing the productive energy content, lowering the protein level, and careful amino
AMINO ACID SUPPLEMENTATION OF FOODS AND FEEDS 4 1 5
acid supplementation (Baldini et al, 1957). This may become the first practical use of two amino acids when the price of lysine should make this more economically attractive.
3. Pig Diets
Catron et al. (1953) have published the results of a study in which pigs were given a 12% corn-soybean oil meal diet (Table V) supple-mented with graded doses of lysine. The results, shown in Table IX,
TABLE IX
SUPPLEMENTATION OF PIG D I E T WITH L-LYSINE-HCI®
L-Lysine · HCl as supplement
to diet (%) 0 0.05 0.10 0.15
Daily gain (pounds)
1.07 1.11 1.21 1.18
Feed Gain 3.74 3.22 3.01 3.14
a Poland China X Landrace X Durve pigs self-fed from an average of 22.8 pounds to 100 pounds.
illustrate the point made earlier that corn-soybean oil meal mixtures tend to become deficient in lysine as the protein level is lowered. Best response was obtained with 0.1% supplementary L-lysine · HCl. These data are of considerable practical importance as many farmers in the corn belt feed their pigs probably no better diets than the unsupple-mented diet used in this test.
VI. CONCLUSION
It has been shown that the amount of "effective," "balanced," or "com-plete" protein in any food or feed can be increased by appropriate sup-plementation with the first limiting amino acid. The amount of supple-mentation that can be used effectively is governed mainly by the con-centration of the second limiting amino acid present in the food or feed and available to the organism. Proper supplementation is achieved when the amount of the first is in balance with the amount of the second lim-iting amino acid and with the rest of the protein according to the needs of the species. All other nutrients must, of course, be present in the diet to assure full utilization of the balanced portion of the protein. These theoretically sound principles are used presently by the highly com-petitive feed industry for formulation of various poultry diets. Little use has been made of the opportunities for the human dietary with the ex-ception of providing a superior bread which is commercially available in some areas of this country. Further development awaits the demon-stration that the improvement of basic foodstuffs such as rice and corn
416 HANS R. ROSENBERG
can be of benefit. As these data become available amino acid supple-mentation will be used wherever needed to improve the health and well-being of people throughout the world.
REFERENCES
Albanese, A. A., Higgons, R. A., Hyde, G. M., and Orto, L. (1955a). N.Y. State J. Med. 55, 3453.
Albanese, A. A., Higgons, R. A., Hyde, G. M., and Orto, L. (1955b). Am. J.
Clin. Nutrition 3, 121.
Albanese, A. A., Higgons, R. A., Hyde, G. M., and Orto, L. (1956). Am. J. Clin.
Nutrition 4, 161.
Albanese, A. A., Higgons, R. A., Orto, L., and Zavattaro, D. N. (1957). Geriatrics 12, 465.
Allison, J. B. (1949). Advances in Protein Chem. 1, 155-200.
Allison, J. B. (1951). Federation Proc. 10, 676.
Allison, J. B. (1955). Physiol. Revs. 35, 664.
Allison,}. B. (1957). /. Am. Med. Assoc. 164, 283.
Baldini, J. T. (1958). Proc, llth World Poultry Set. Congr. Mexico City. (In press.)
Baldini, J. T., and Rosenberg, H. R. (1955). Poultry Sei. 34, 1301.
Baldini, J. T., and Rosenberg, H. R. (1957). Poultry Sei. 36, 432.
Baldini, J. T., Rosenberg, H. R., and Waddell, J. (1954). Poultry Sei. 33, 539.
Baldini, J. T., Marvel, J. P., and Rosenberg, H. R. (1957). Poultry Set. 36, 1031-1035.
Bender, A. E. (1958). Science 127, 874.
Bender, A. E., and Doell, B. H. (1957). Brit. J. Nutrition 11, 140-148.
Bird, H. R. (1955). Poultry Set. 34, 1163.
Block, R. J., and Weiss, K. W. (1956). "Amino Acid Handbook." C. C Thomas, Springfield, Illinois.
Catron, D. V., Acker, D. C., Ashton, G. C., Maddock, H. M., and Speer, V. C.
(1953). /. Animal Set. 12, 910.
Chapman, D. G., Castillo, R., and Campbell, J. A. (1959). Can. J. Biochem.
Physiol. 37, 679.
Culik, R., and Rosenberg, H. R. (1958). Food Technol. 12, 169.
Derse, P. H. (1958). /. Assoc. Offic. Agr. Chemists 41, 192.
Deshpande, P. D., Harper, A. E., Collins, M., and Elvehjem, C. A. (1957). Arch.
Biochem. Biophys. 67, 341.
Edwards, C. H., and Allen, C. H. (1958). /. Agr. Food Chem. 6, 219.
Fisher, H., Dowling, J., and Maddy, K. H. (1956). Poultry Set. 35, 239.
Flodin, N. W. (1953). /. Agr. Food Chem. 1, 222.
Flodin, N. W. (1957). Metabolism Clin. and Exptl. 6, 360.
Food and Agr. Organization U.N., FAO Nutritional Studies (1957) No. 16 (Protein Requirements).
Fraps, G. S. (1946). Texas Agr. Expt. Sta. Bull. No. 678.
Gupta, J. D., and Elvehjem, C. A. (1957). /. Nutrition 62, 313.
Gupta, J. D., Dakroury, A. M., Harper, A. E., and Elvehjem, C. A. (1958). /.
Nutrition 64, 259.
Harper, A. E., and Elvehjem, C. A. (1957). /. Agr. Food Chem. 5, 754.
Howard, H. W., Monson, W. J., Bauer, C. D., and Block, R. J. (1958). /. Nutri-tion 64, 151.
AMINO ACID SUPPLEMENTATION OF FOODS AND FEEDS 4 1 7 Howe, E. E. (1958). Borden s Rev. Nutrition Research 19, 19.
Hutchinson, J. B., Moran, T., and Pace, J. (1956). Proc. Roy. Soc. B146, 270.
Jahnke, J. K., and Schuck, C. (1957). /. Nutrition 61, 307.
Lantz, E. L., and Wood, P. (1958). /. Am. Dietet. Assoc. 34, 138.
Longenecker, J. B., and Hause, N. L. (1958). Nature 182, 1739.
McCollum, E. V., and Simmonds, N. (1929). "The Newer Knowledge of Nutri-tion," 4th ed. Macmillan, New York.
Mack, P. B. (1956). Abstr. of Papers, 129th Meeting, Am. Chem. Soc, Dallas, Texas, p. 12 C.
Miller, D. S., and Bender, A. E. (1955). Brit. J. Nutrition 9, 382.
Mitchell, H. H. (1944). Ind. Eng. Chem. Anal. Ed. 16, 696.
Mitchell, H. H., and Block, R. J. (1946). /. Biol. Chem. 163, 599.
Mitchell, H. H., and Smuts, D. B. (1932). /. Biol. Chem. 95, 263.
Osborne, T. B., and Mendel, L. B. (1919). /. Biol. Chem. 37, 223 and 557.
Osborne, T. B., and Mendel, L. B. (1920). /. Biol. Chem. 41, 275.
Pecora, L. J., and Hundley, J. M. (1951). /. Nutrition 44, 101.
Rose, W. C. (1937). Science 86, 298.
Rose, W. C. (1938). Physiol. Revs. 18, 109.
Rose, W. C. (1957). Nutrition Ahstr. b- Revs. 27, 631.
Rose, W. C, and Rice, E. E. (1939). Science 90, 186.
Rose, W. C, Smith, L. C , Womack, M., and Shane, M. (1949). J. Biol. Chem.
181, 307.
Rosenberg, H. R. (1957). J. Agr. Food Chem. 5, 694-700.
Rosenberg, H. R. (1959). /. Agr. Food Chem. 7, 316.
Rosenberg, H. R., and Baldini, J. T. (1957). Poultry Sei. 36, 247-252.
Rosenberg, H. R., and Culik, R. (1955). /. Animal Sei. 14, 1221.
Rosenberg, H. R., and Culik, R. (1957). /. Nutrition 63, 477.
Rosenberg, H. R., and Rohdenburg, E. L. (1951). /. Nutrition 45, 593.
Rosenberg, H. R., and Rohdenburg, E. L. (1952). Arch. Biochem. Biophys. 37, 461.
Rosenberg, H. R., Baldini, J. T., Sunde, M. L., Bird, H. R., and Runnels, T. D.
(1955). Poultry Set. 34, 1308-1313.
Rosenberg, H. R., Baldini, J. T., and Tollefson, C. I. (1957). Poultry Sei. 36, 1381-1382.
Rosenberg, H. R., Culik, R., and Eckert, R. E. (1959). Federation Proc. 18, 544.
Sabiston, A., and Kennedy, B. (1957). Cereal Chem. 34, 94.
Säuberlich, H. E., Chang, Wan-Yuin, and Salmon, W. D. (1953). J. Nutrition 51, 623.
Schwartz, H. G., Taylor, M. W., and Fisher, H. (1958). /. Nutrition 65, 25-37.
Scrimshaw, N. S., Arroyave, G., and Bressani, R. (1958). Ann. Rev. Biochem.
27, 403-426.
Steams, G., Newman, K. J., McKinley, J. B., and Jeans, P. C. (1958). Ann. Ν.Ύ.
Acad. Set. 69, 857.
Stephenson, E. L. (1956). Feed Age 6, 39.
Stephenson, E. L. (1956). Feed Age 6, 39.