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Determination of Limiting Amino Acids of Rumen-Isolated Microbial Proteins Fed to Rat1
TECHNICAL
NOTES
Determination of Limiting Amino Acids of Rumen-lsolated Microbial Proteins Fed to Rat 1 Abstract
The limiting amino acids of rumen microbial proteins were determined with rats using the plasma amino acid score method. Results showed that for rumen protozoal protein, histidine was the first limiting amino acid; cystine was the first limiting amino acid for rumen bacterial protein. Recent work in our laboratory (8, 19) has dealt with the effect of dietary factors on plasma amino acid levels in ruminants, and Purser et al. (19) have suggested a method for limiting amino acid determinations, using essentially a modification of the plasma amino acid score method of McLaughlan (12). A p p l y i n g this method, Klopfenstein et al. (8) suggested that in defaunated shetp lysine was the limiting amino acid of the composite protein reaching the lower gut, whereas in faunated sheep no single essential amino acid was found to be consistently limiting• Feeding trials (5, 6, 9-11) or amino acid analysis of abomasal hydrolysates (17) have suggested that methionine or lysine may be the limiting amino acids of the composite protein reaching the lower gut of ruminants; however, these data can not be interpreted unequivocally, since the feeding trial data could not establish an effect of amino acid supplementation per se and it is well realized that amino acid compositions of proteins are not necessarily indicative of amino acid availability (4). While it is realized that the protein presented to the ruminant for digestion may not be solely of microbial origin, it was considered desirable to determine the limiting amino acids of rumen microbial protein using the McLaughlan score method (12) to facilitate interpretation of the data obtained with ruminant animals. Experimental Procedure
well, and the rumen protozoa and bacteria isolated by differential centrifugation. The protozoa were removed after centri_fugation at 150 × g for 10 rain; the 150 × g supernatant was recentrifuged at 20,000 × g for 10 rain to remove bacteria. The microbial preparations were then spread on a glass plate, dried in a forced-air oven at 39 C, collected, and finally stored at 4 C. The rumen contents were collected two or three times weekly over a period of several months till about 450 g of dry rumen protozoa and 400 g of dry rumen bacteria had been collected. The plasma amino acid score ( P A A - S ) method of McLaughlan (12) and the restricted feeding regimen of rats with 10% protein rations of McLaughlan et al. (13) were used to determine limiting amino acids of the rumen microbial protein sources. Blood samples were drawn from the rats by heart puncture and plasma amino acids ( P A A ) determined as described by Purser et al. (,19). To compare the rumen microbial preparations to those fed previously to rats (14, 15) the biological values of these rumen microbial preparations were determined according to the ThomasMitchell procedure. Nitrogen analyses were done according to the Kjeldahl procedure, crude fiber anMysis according to conventional proximate analysis methods (1), and the amino acid compositions of the microbial preparations analyzed as described (2). Results and Discussion
Microscopic and chemical examination (crude fiber determination) indicated that the two microbial preparations were practically devoid of feed or plant material contamination. The crude fiber content was 1.5 and 0.7% for the rumen protozoal and bacterial preparations, respectively. Assuming that at 4 hr post-feeding the crude fiber represented about 60% of the residual feed contamination, and the rest of the residual feed contamination represented purely protein, only 0.8 and 0.4%, respectively, of the protein in the protozoal and bacterial preparations were of nonmicrobial origin. Table 1 lists the bulk amino acid compositions of the two rumen microbial preparations. These data are similar to those reported previously (18), but threonine, methionine, and leucine were somewhat higher and lysine somewhat lower in the bacterial preparation. I n the protozoal preparation threonine was higher and valine and lysine were somewhat lower than previously indicated. Most of the differences between the
Six rumen-fistulated sheep (average body weight 42 kg) were fed a basal ration containing 44% alfalfa meal, 39.2% ground shelled corn, 5% ground corn cobs, 5% molasses, 4.8% cura-phosphate, limestone, trace-mineral salt mixture, and 2% urea twice daily at 7% of their metabolic body weight ( B W ~ ) . Rumen contents were collected from each sheep 4 hr after the morning feed, combined and squeezed through two layers of cheese cloth. The fluid portion was diluted 1:1 (v/v) with phosphate buffer ( p H 6.5) and incubated for 1 hr at 39 C in Bunsen valve-equipped separatory funnels. The feed-residue-free, lower fluid portion and the settled large protozoa were subsequently 1 Supported in part by NIH Training Grant withdrawn from the separatory funnels, mixed TIES17 and Eli Lilly and Company. 1698
TECHSICAL ~OTES TABLE 1. Bulk amino acid composition of the rumen microbial preparations. ~ Amino Acid Aspartic Acid Threonine Serine Glutamic Acid Proline Glycine Alanine Valine Methionine Isoleucine Leuclne Tyrosine Phenylalanine Lysine Histidine Arginine
Rumen bacteria b
Rumen protozoa ~
11.1 7.3 4.3 8.8 2.3 4.0 9.6 6.6 3.6 6.0 8.1 5.4 6.2 8.0 2.4 5.2
14.6 5.7 4.5 13.5 3.3 5.4 4.7 4.2 2.5 6.7 8.5 5.2 5.7 9.0 2.4 4.0
Per cent distribution by weight (grams amino acid ~er 100 g amino acid). b Crude protein content was 35.3%. Crude protein content was 50.1%. amino acid compositions of the bacterial preparations from present and previous studies were within standard deviations published (18) for the average amino acid composition of 22 rumen bacterial strains. Results of the Biological Value (BV), True Digestibility (TD), and Net Protein Utilization (NPU) determinations are given in Table 2. The BV were 85 and 82 for rumen bacteria and protozoa, respectively, and the TD of the tureen protozoa was higher than that of the rumen bacteria; thus, the N P U of rumen protozoa exceeded that of the rmnen bacteria. These data agree well with reported results (7, 14, 15). Results for the limiting amino acid determinations are given in Table 3. F o r tureen DrotozoM protein, histidine was predicted as the limiting amino acid, whereas eystine, arginine; valine, leucine, and threonine were indicated as the next five least available amino acids. Lysine was not among these less available amino acids of tureen protozoal protein. This finding agreed well with work by Klopfenstein et al. (8), who showed that in faunated sheep lysine was not the predominant limiting amino acid. t to w e v e r , no work has been reported indicating histidine as the most limiting amino acid of tureen protozoal
1699
protein. The plasma concentrations of free histitidine in rats fed protozoal protein-containing diets for ten days were extremely low (3), indicating that this acid was limiting. The result is of interest, for histidine has b e e n noted as being in greater relative concentrations in the plasma when steers were fed diets detrimental to protozoa (16). F o r rumen bacterial protein, cystine was predicted as the limiting amino acid, whereas arginine, histidine, leucine, and lysine were indicated as the next four least available amino acids. Poley (17) had concluded that the sulfurcontaining amino acids were the limiting ones in ruminants. However, Klopfenstein et al. (8) indicated that lysine was the most limiting amino acid in defaunated sheep, which might have been influenced by the high quantity of corn, which is usually limiting in lysine, in the diet (as already indicated). The results indicated the following conclusions: The limiting amino acid data from ratfeeding trials of rumen microbial protein sources were similar to previous, more direct limiting amino acid determinations in ruminants (8, 17, 19). These similarities probably reflected the general quantitative importance of microbial proteins in the composite protein digested in the lower gut of ruminants. F o r meaningful analysis of these results, cognizance must be taken that in rats, rumen microbial protein was the sole dietary protein source, whereas the protein digested in the lower gut of ruminants may be of microbial as well as of feed origin. W . G. BERGEN, 2 D. B. PURSER, ~ and J. H. CLINE
Institute of Nutrition and Department of Animal Science The Ohio State University, Columbus
~Present address: Animal Husbandry Department, Michigan Sta~e University, East Lansing. References
(1) Association of Official Agricultural Chemists. 1960. Methods of Analysis. 9th ed. Washington, D.C. (2) Bergen, W. G., D. B. Purser, and J. H. Cline. 1967. Enzymatic dete~nination of the protein quality of individual tureen bacteria. J. Nutrition, 92: 357. (3) Bergen, W. G., and D. B. Purser. 1968. The effect of feeding different protein sources on plasma and gut amino acids in the growing rat. J. Nutrition, 95: 333.
T~LE 2. Biological value, true digestibility, and net protein utilization of rumen bacteria and rumen protozoa. Protein source
Protein % in diet
Rumen bacteria Rumen protozoa Casein ~
9 9 9
BV
TD
NPU
85.0 ___0.6" 82.0 +_ 2.1 89.5 ± 0.5
74.6 ± 0.8 87.2 ± 1.2 97.2 +__0.5
63.4 (7) b 71.4 (7) 87.0 (6)
a Mean ----_SE. b Number of rats in each mean. c Positive control. J. DAIRY SCIENCE VOL. 51. NO. 10
1700
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(4) Carpenter, K. J., B. E. March, C. K. Milner, and R. C. Campbell. 1963. A growth assay with chicks for the lysi.ae content of protein concentrates. Brit. J. Nutrition, 17 : 309. (5) Gosset, W. It., T. W. Perry, M. T. Mohler, M. P. Plumlee, and W. M. Beeson. 1962. Value of supplemental lysine, methionlne, methionine analog and trace minerals on high urea f a t t e n i n g rations for steers. J. Anim. Sci., 21: 248. (6) ttarbers, L. H., R. R. Oltjen, and A. D. Tillman. 1961. Lysine supplementation in rations for sheep. J . Anita. Sci., 20: 880. (7) IIungate, R. E. 1966. The Rumen and I t s Microbes. Academic Press, New York. (8) Klopfenstein, T. ft., D. B. Purser, and W. J. Tyznik. 1966. Effects of defaunation on feed digestibility, rumea metabolism and blood metabolites. J . Anita. Sci., 25: 765. (9) Lofgreen, G. P., J. K. Loosli, and L. A. Maynard. 1947. The influence of protein source upon nitrogen retention by sheep. J. Anim. Sci., 6: 343. (10) Loosli, J. K., and L. E. Harris. 1945. Methionine increases the value of urea for lambs. J. Anim. Sci., 4: 435. (11) MeLaren, G. A., G. L. Anderson, and K. M. Barth. 1965. Influence of methionine and tryptophan on nitrogen utilization by lambs fed high levels of non-proteln nitrogen. J. Anita. Sci., 24: 231. (12) MeLaughlan, J. M. ]964. Blood amino acid studies. 5. Determination of the limiting amino acid in diets. Canadian J. Biochem., 42: 1353. (13) McLaughlan, J. M., S. V. Rao, F. Y. Noel, and A. B. Morrisom 1967. Blood amino acid studies. 7. Use of plasma amino acid score for predicting limiting amino acid(s) in dietary proteins. Canadian J. Biochem., 45 : 3]. (14) MeNaught, M. L., A. B. Smith, K. M. Henry, and S. K. Kon. ]950. The utilization of non-protein nitrogen in the bovine tureen. 5. The isolation and nutritive value of a preparation of dried tureen bacteria. Bio: chem. J., 46: 32. (15) MeNaught, M. L., E. C. Owen, K. M. Henry, and S. K. Ken. 1954. The utilization of non-protein nitrogen in the bovine rumen. 8. The nutritive value of the proteins of preparations of dried rumen bacteria, rumen protozoa and brewer's yeast for rats. Biochem. J., 56: 151. (16) Oltjen, R. R., and P. A. Putnam. 1966. Plasma amino acids and nitrogen retention by steers fed purified diets containing urea or isolated soy protein. J. Nutrition, 89 : 385. (17) Poley, O. E. 1965. Influence of dietary nitrogen sources on amino acids in plasma and abomasa] ingesta from sheep. Ph.D. thesis, Iowa State University, Ames, Iowa. (18) Purser, D. B., and S. M. Buechler. 1966. Amino acid composition of rumen organisms. J. Dairy Sei., 49: 81. (19) Purser, D. B., T. J. }~lopfenstein, and J. H. Cline. 1966. Dietary and defaunation effects upon plasma amino acid concentrations in sheep. J. Nutrition, 89: 226.
NOTES
Determination of Limiting Amino Acids of Rumen-lsolated Microbial Proteins Fed to Rat 1 Abstract
The limiting amino acids of rumen microbial proteins were determined with rats using the plasma amino acid score method. Results showed that for rumen protozoal protein, histidine was the first limiting amino acid; cystine was the first limiting amino acid for rumen bacterial protein. Recent work in our laboratory (8, 19) has dealt with the effect of dietary factors on plasma amino acid levels in ruminants, and Purser et al. (19) have suggested a method for limiting amino acid determinations, using essentially a modification of the plasma amino acid score method of McLaughlan (12). A p p l y i n g this method, Klopfenstein et al. (8) suggested that in defaunated shetp lysine was the limiting amino acid of the composite protein reaching the lower gut, whereas in faunated sheep no single essential amino acid was found to be consistently limiting• Feeding trials (5, 6, 9-11) or amino acid analysis of abomasal hydrolysates (17) have suggested that methionine or lysine may be the limiting amino acids of the composite protein reaching the lower gut of ruminants; however, these data can not be interpreted unequivocally, since the feeding trial data could not establish an effect of amino acid supplementation per se and it is well realized that amino acid compositions of proteins are not necessarily indicative of amino acid availability (4). While it is realized that the protein presented to the ruminant for digestion may not be solely of microbial origin, it was considered desirable to determine the limiting amino acids of rumen microbial protein using the McLaughlan score method (12) to facilitate interpretation of the data obtained with ruminant animals. Experimental Procedure
well, and the rumen protozoa and bacteria isolated by differential centrifugation. The protozoa were removed after centri_fugation at 150 × g for 10 rain; the 150 × g supernatant was recentrifuged at 20,000 × g for 10 rain to remove bacteria. The microbial preparations were then spread on a glass plate, dried in a forced-air oven at 39 C, collected, and finally stored at 4 C. The rumen contents were collected two or three times weekly over a period of several months till about 450 g of dry rumen protozoa and 400 g of dry rumen bacteria had been collected. The plasma amino acid score ( P A A - S ) method of McLaughlan (12) and the restricted feeding regimen of rats with 10% protein rations of McLaughlan et al. (13) were used to determine limiting amino acids of the rumen microbial protein sources. Blood samples were drawn from the rats by heart puncture and plasma amino acids ( P A A ) determined as described by Purser et al. (,19). To compare the rumen microbial preparations to those fed previously to rats (14, 15) the biological values of these rumen microbial preparations were determined according to the ThomasMitchell procedure. Nitrogen analyses were done according to the Kjeldahl procedure, crude fiber anMysis according to conventional proximate analysis methods (1), and the amino acid compositions of the microbial preparations analyzed as described (2). Results and Discussion
Microscopic and chemical examination (crude fiber determination) indicated that the two microbial preparations were practically devoid of feed or plant material contamination. The crude fiber content was 1.5 and 0.7% for the rumen protozoal and bacterial preparations, respectively. Assuming that at 4 hr post-feeding the crude fiber represented about 60% of the residual feed contamination, and the rest of the residual feed contamination represented purely protein, only 0.8 and 0.4%, respectively, of the protein in the protozoal and bacterial preparations were of nonmicrobial origin. Table 1 lists the bulk amino acid compositions of the two rumen microbial preparations. These data are similar to those reported previously (18), but threonine, methionine, and leucine were somewhat higher and lysine somewhat lower in the bacterial preparation. I n the protozoal preparation threonine was higher and valine and lysine were somewhat lower than previously indicated. Most of the differences between the
Six rumen-fistulated sheep (average body weight 42 kg) were fed a basal ration containing 44% alfalfa meal, 39.2% ground shelled corn, 5% ground corn cobs, 5% molasses, 4.8% cura-phosphate, limestone, trace-mineral salt mixture, and 2% urea twice daily at 7% of their metabolic body weight ( B W ~ ) . Rumen contents were collected from each sheep 4 hr after the morning feed, combined and squeezed through two layers of cheese cloth. The fluid portion was diluted 1:1 (v/v) with phosphate buffer ( p H 6.5) and incubated for 1 hr at 39 C in Bunsen valve-equipped separatory funnels. The feed-residue-free, lower fluid portion and the settled large protozoa were subsequently 1 Supported in part by NIH Training Grant withdrawn from the separatory funnels, mixed TIES17 and Eli Lilly and Company. 1698
TECHSICAL ~OTES TABLE 1. Bulk amino acid composition of the rumen microbial preparations. ~ Amino Acid Aspartic Acid Threonine Serine Glutamic Acid Proline Glycine Alanine Valine Methionine Isoleucine Leuclne Tyrosine Phenylalanine Lysine Histidine Arginine
Rumen bacteria b
Rumen protozoa ~
11.1 7.3 4.3 8.8 2.3 4.0 9.6 6.6 3.6 6.0 8.1 5.4 6.2 8.0 2.4 5.2
14.6 5.7 4.5 13.5 3.3 5.4 4.7 4.2 2.5 6.7 8.5 5.2 5.7 9.0 2.4 4.0
Per cent distribution by weight (grams amino acid ~er 100 g amino acid). b Crude protein content was 35.3%. Crude protein content was 50.1%. amino acid compositions of the bacterial preparations from present and previous studies were within standard deviations published (18) for the average amino acid composition of 22 rumen bacterial strains. Results of the Biological Value (BV), True Digestibility (TD), and Net Protein Utilization (NPU) determinations are given in Table 2. The BV were 85 and 82 for rumen bacteria and protozoa, respectively, and the TD of the tureen protozoa was higher than that of the rumen bacteria; thus, the N P U of rumen protozoa exceeded that of the rmnen bacteria. These data agree well with reported results (7, 14, 15). Results for the limiting amino acid determinations are given in Table 3. F o r tureen DrotozoM protein, histidine was predicted as the limiting amino acid, whereas eystine, arginine; valine, leucine, and threonine were indicated as the next five least available amino acids. Lysine was not among these less available amino acids of tureen protozoal protein. This finding agreed well with work by Klopfenstein et al. (8), who showed that in faunated sheep lysine was not the predominant limiting amino acid. t to w e v e r , no work has been reported indicating histidine as the most limiting amino acid of tureen protozoal
1699
protein. The plasma concentrations of free histitidine in rats fed protozoal protein-containing diets for ten days were extremely low (3), indicating that this acid was limiting. The result is of interest, for histidine has b e e n noted as being in greater relative concentrations in the plasma when steers were fed diets detrimental to protozoa (16). F o r rumen bacterial protein, cystine was predicted as the limiting amino acid, whereas arginine, histidine, leucine, and lysine were indicated as the next four least available amino acids. Poley (17) had concluded that the sulfurcontaining amino acids were the limiting ones in ruminants. However, Klopfenstein et al. (8) indicated that lysine was the most limiting amino acid in defaunated sheep, which might have been influenced by the high quantity of corn, which is usually limiting in lysine, in the diet (as already indicated). The results indicated the following conclusions: The limiting amino acid data from ratfeeding trials of rumen microbial protein sources were similar to previous, more direct limiting amino acid determinations in ruminants (8, 17, 19). These similarities probably reflected the general quantitative importance of microbial proteins in the composite protein digested in the lower gut of ruminants. F o r meaningful analysis of these results, cognizance must be taken that in rats, rumen microbial protein was the sole dietary protein source, whereas the protein digested in the lower gut of ruminants may be of microbial as well as of feed origin. W . G. BERGEN, 2 D. B. PURSER, ~ and J. H. CLINE
Institute of Nutrition and Department of Animal Science The Ohio State University, Columbus
~Present address: Animal Husbandry Department, Michigan Sta~e University, East Lansing. References
(1) Association of Official Agricultural Chemists. 1960. Methods of Analysis. 9th ed. Washington, D.C. (2) Bergen, W. G., D. B. Purser, and J. H. Cline. 1967. Enzymatic dete~nination of the protein quality of individual tureen bacteria. J. Nutrition, 92: 357. (3) Bergen, W. G., and D. B. Purser. 1968. The effect of feeding different protein sources on plasma and gut amino acids in the growing rat. J. Nutrition, 95: 333.
T~LE 2. Biological value, true digestibility, and net protein utilization of rumen bacteria and rumen protozoa. Protein source
Protein % in diet
Rumen bacteria Rumen protozoa Casein ~
9 9 9
BV
TD
NPU
85.0 ___0.6" 82.0 +_ 2.1 89.5 ± 0.5
74.6 ± 0.8 87.2 ± 1.2 97.2 +__0.5
63.4 (7) b 71.4 (7) 87.0 (6)
a Mean ----_SE. b Number of rats in each mean. c Positive control. J. DAIRY SCIENCE VOL. 51. NO. 10
1700
J O U R N A L O F DAII%Y S C I E N C E
i
{5 +, +, +, +I+i +, +, +I+, +I+I
o
v O
_~~ ~
÷t +I +I +I +I +I +I +I +I +I +I
.~ ~ -~
4-1+1 +1 +1 +1 +1 +1 +1 +1 +1 ÷1
~
+l +l +l +I +] +I H +t 41+l +l
O
,m
gh¢2
.~
b~
~
g
4141+I+141+t4141414141
g
J . DAIR~ S c I E n C E VOL. 51, NO. 1 0
~
(4) Carpenter, K. J., B. E. March, C. K. Milner, and R. C. Campbell. 1963. A growth assay with chicks for the lysi.ae content of protein concentrates. Brit. J. Nutrition, 17 : 309. (5) Gosset, W. It., T. W. Perry, M. T. Mohler, M. P. Plumlee, and W. M. Beeson. 1962. Value of supplemental lysine, methionlne, methionine analog and trace minerals on high urea f a t t e n i n g rations for steers. J. Anim. Sci., 21: 248. (6) ttarbers, L. H., R. R. Oltjen, and A. D. Tillman. 1961. Lysine supplementation in rations for sheep. J . Anita. Sci., 20: 880. (7) IIungate, R. E. 1966. The Rumen and I t s Microbes. Academic Press, New York. (8) Klopfenstein, T. ft., D. B. Purser, and W. J. Tyznik. 1966. Effects of defaunation on feed digestibility, rumea metabolism and blood metabolites. J . Anita. Sci., 25: 765. (9) Lofgreen, G. P., J. K. Loosli, and L. A. Maynard. 1947. The influence of protein source upon nitrogen retention by sheep. J. Anim. Sci., 6: 343. (10) Loosli, J. K., and L. E. Harris. 1945. Methionine increases the value of urea for lambs. J. Anim. Sci., 4: 435. (11) MeLaren, G. A., G. L. Anderson, and K. M. Barth. 1965. Influence of methionine and tryptophan on nitrogen utilization by lambs fed high levels of non-proteln nitrogen. J. Anita. Sci., 24: 231. (12) MeLaughlan, J. M. ]964. Blood amino acid studies. 5. Determination of the limiting amino acid in diets. Canadian J. Biochem., 42: 1353. (13) McLaughlan, J. M., S. V. Rao, F. Y. Noel, and A. B. Morrisom 1967. Blood amino acid studies. 7. Use of plasma amino acid score for predicting limiting amino acid(s) in dietary proteins. Canadian J. Biochem., 45 : 3]. (14) MeNaught, M. L., A. B. Smith, K. M. Henry, and S. K. Kon. ]950. The utilization of non-protein nitrogen in the bovine tureen. 5. The isolation and nutritive value of a preparation of dried tureen bacteria. Bio: chem. J., 46: 32. (15) MeNaught, M. L., E. C. Owen, K. M. Henry, and S. K. Ken. 1954. The utilization of non-protein nitrogen in the bovine rumen. 8. The nutritive value of the proteins of preparations of dried rumen bacteria, rumen protozoa and brewer's yeast for rats. Biochem. J., 56: 151. (16) Oltjen, R. R., and P. A. Putnam. 1966. Plasma amino acids and nitrogen retention by steers fed purified diets containing urea or isolated soy protein. J. Nutrition, 89 : 385. (17) Poley, O. E. 1965. Influence of dietary nitrogen sources on amino acids in plasma and abomasa] ingesta from sheep. Ph.D. thesis, Iowa State University, Ames, Iowa. (18) Purser, D. B., and S. M. Buechler. 1966. Amino acid composition of rumen organisms. J. Dairy Sei., 49: 81. (19) Purser, D. B., T. J. }~lopfenstein, and J. H. Cline. 1966. Dietary and defaunation effects upon plasma amino acid concentrations in sheep. J. Nutrition, 89: 226.