D E G R A D A T I O N OF A MI N O ACIDS BY PURE CULTURES OF RUMEN BACTERIA Curtis Scheifinger 1 , Neville Russell and William Chalupa /
SUMMARY
Amino Acid (AA) degradation profiles of five major genera of rumen bacteria were determined using physiological levels of AA under in vitro conditions. The results indicated that (1) not all AA are degraded by all strains of rumen bacteria and (2) degradation occurs at different rates. The genera Megaspbaera, Eubacterium and Streptococcus isolate 19D degraded all 14 AA tested. Members of the genus Butyrivibrio totally degraded Ser, Asp and Glu, with Gly being the only AA not attacked. The two subspecies of Selenomonas tested, differed in their AA degradation patterns. Subspecies lactilytica degraded all AA tested except His and Tyr. Subspecies ruminantium Could not degrade Gly, Leu, lie, Thr, His, Arg, Lys, Trp nor Tyr. Met appeared unique in that it was produced by members of Megaspbaera, Eubacterium and isolate 19D of Streptococcus. Selenomonas, Butyrivibrio and Streptococcus isolated 12D, however, degraded Met. It thus appears that the total ruminal degradation of dietary AA occurs as a result of extensive bacterial interaction. (Key Words: Amino Acid, Degradation, Rumen Bacteria.)
proteolysis. Bacteria involved have been isolated, identified and the proteolytic enzymes studied (Warner, 1956; Hunt and Moore, 1958; Blackburn and Hobson, 1960a,b; Fulghum and Moore, 1963; Borchers, 1965). The fate of free amino acids (AA) has been studied only in experiments designed primarily for the study of proteolysis. In many of these AA catabolism studies, work was performed using high substrate (AA) concentrations and washed or fractionated cell suspensions. Deamination was measured as a function of NH3 production, thus not accounting for those AA utilized by bacterial uptake, binding or decarboxylation. The investigation of AA catabolism involved isolating pure cultures of rumen bacteria that have the ability to use AA for energy and then using these isolates as tools to study AA degradation. Disappearance of 14 AA was measured as a function of microbial growth to obtain results to refute or support a general type deaminase system shared by all bacteria. MATERIALS AND METHODS
Isolation. Samples of rumen fluid were obtained from a 500 kg fistulated steer fed 2 x/day a 16% protein diet consisting of 10 kg alfalfa hay (N.R.C. 1-00-022) and 1 kg Agway Campion Concentrate4 ; water was available ad INTRODUCTION libitum. The animal was maintained on this diet In recent years, much investigative effort has for several months prior to the study. The been directed towards understanding rumen animal was fed at 0800 and 1500 hr and was sampled 2 hr post 0800 feeding. Samples were 1Present address: Eli Lilly and Co., Greenfield strained through one layer of cheesecloth into a Labs., Greenfield, IN 46140. 2,000 ml Erlenmeyer flask warmed to 39 C and 2Present address: Dept. of Dairy Science, Univer- flushed with O2-free CO2 obtained by passing sity of Maryland, College Park, 20740. CO2 through heated copper filings to remove 3Applebrook Research Center, SmithKline Animal any 02. Health Products, 1600 Paoli Pike, West Chester, PA Dilutions were made in the anaerobic dilu19380. 4Agway Inc., Syracuse, NY. Grain products, plant tion fluid of Bryant (1961) and the anaerobic protein products, grain by-products, calcium propioculture techniques of Hungate (1966) and hate, cane molasses, limestone, nono-calcium phosBryant (1972) were employed throughout the phates, dicalcium phosphate, salt, cobalt sulfate, copper sulfate, zinc sulfate, magnesium oxide, vita- study. mins A and E. Bacteria were isolated from roll tubes con821 JOURNAL OF ANIMAL SCIENCE, Vol. 43, No. 4 (1976)
Downloaded from https://academic.oup.com/jas/article-abstract/43/4/821/4697580 by Iowa State University user on 17 January 2019
SrnitbKline Corp. 3, West Cbester, Pennsylvania 19380
822
SCHEIFINGER, RUSSELL AND CHALUPA
s Technicon TSM Amino Acid Analyzer, Technicon Corporation, Tarrytown, NY.
KH2PO4, .6% (NH4)2 SO4, 1.2% NaCI, .24% MgSO4 " 7 H20, .16% CaCI2 9 2 H20) and .1 ml of .1% reaszurin. The following L-amino acids were added to yield a final concentration of 15 mM each: Ala, Arg 9 HC1, Asp, Glu, Gly, His 9 HC1, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp and Val. Tyrosine was added at 10 millimoles. The pH was adjusted to 6.7 and the medium was autoclaved for 15 minutes at 15 lb/in 2 under CO2 in a sealed flask. After cooling to 50 C, 5 ml of sterile 8% Na2 COa and 2 ml of sterile 2.5% Cysteine 9 HC1 were added. The agar medium was then dispensed into 18 • 150 mm tubes stoppered with butyl rubber stoppers under O2-free CO2 in 10 ml amounts. The medium for the initial deaminase test was basically the isolation medium devoid of agar, Cys 9 HC1, Pro, Ser, Trp, Glu, Gly, Ala and Asp. Glucose, cellobiose, maltose and soluble starch were added at .1%, respectively, to provide energy for growth. The amino acid analysis medium was that of the isolation medium except agar and Cys " HCI were deleted, the AA concentration was 1.5 mM and glucose, cellobiose, maltose and soluble starch were added at .1%, respectively, to provide energy. R ESU LTS
Bacteria from the initial isolation were tested for their ability to deaminate a mixture of 10 AA that are considered to be essential for growth and maintenance at the ruminant tissue level, Downes (1961). Organisms giving ~20% deamination based on the decrease in total c~ amino-N were saved and catagorized into genera on the basis of their gross morphological characteristics as shown in table 1. The Selenomonads were further classified into subspecies based on their abilities to grow on lactate and glycerol. The fermentation characteristics of the other isolates supplemented the gross morphological classification into genera. The organisms isolated represent genera to be active in rumen proteolysis (Bladen et al. 1961). Table 2 shows the results of the initial deaminase test. S. ruminantium subsp, lactilytica gave the greatest decrease in c~ amino-N while subsp, ruminantium exhibited the least degradation. The remaining genera gave approximately similar degradation rates somewhere between the two extremes. The results of this test only suggest an
Downloaded from https://academic.oup.com/jas/article-abstract/43/4/821/4697580 by Iowa State University user on 17 January 2019
taining the agar media of Bryant and Burkey (1953) modified to contain AA instead of carbohydrates as energy sources. All incubations were at 39 C and those colonies developing larger than background pin point were picked and maintained on RGCA slants as described by Bryant and Robinson (1961). The isolates were tentatively classified into genera on the basis of their morphology. Carbohydrate fermentation characteristics were also determined and used primarily to distinguish the lactate-using and nonlactate-using species of Setenomonas. Culture Growth f o r Amino Acid and Initial Deamination Analysis. The bacteria were grown in 18 • 150 mm tubes containing 10 ml of the appropriate broth under an atmosphere of 02-free CO2. The medium was inoculated from a 24-hr culture using identical broth and growth was allowed to proceed until an optical density 600 nm of approximately .9 was reached (18 hr.). a Amino N Analysis. Two ml of the fermentation broth was centrifuged at 18,000 x g for 30 minutes. To .25 ml of the cell-free supernatant was added 4.5 ml of .5% TCA and 5 ml washed CuPO4 (Borchers, 1959). The ingredients were mixed, cooled for 30 min at 4 C and then centrifuged at 3,000 x g for 10 minutes. This supernatant was used in the a amino-N Cuprizone assay of Borchers (1959) adapted for automated analysis. Results were determined as the decrease in total a amino-N and expressed as the percentage decrease in t~ amino-N from the amount present in the medium before inoculation. Amino Acid Analysis. Fermentation broth was centrifuged at 18,000 x g for 30 minutes. The resulting supernatant was deproteinized with sulfosalicyclic acid and assayed for amino acids, using norleucine as an internal standard with a Technicon amino acid analyzer s. Results are expressed as percentage decrease in the individual AA compared to the amount present in the medium before inoculation. Medium. Isolation media was prepared by first adding the following ingredients and distilled H 2 0 to a final vol of 93 mh .5 g yeast extract, .1 g dithiothreitol, 1.5% agar, 20 ml clarified rumen fluid (Bryant, 1961), 4 ml each of mineral solution 1 (.6% K2 HPO4 ) and 2 (.6%
AMINO ACID DEGRADATION BY PURE CULTURES OF RUMEN BACTERIA
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SUMMARY
Amino Acid (AA) degradation profiles of five major genera of rumen bacteria were determined using physiological levels of AA under in vitro conditions. The results indicated that (1) not all AA are degraded by all strains of rumen bacteria and (2) degradation occurs at different rates. The genera Megaspbaera, Eubacterium and Streptococcus isolate 19D degraded all 14 AA tested. Members of the genus Butyrivibrio totally degraded Ser, Asp and Glu, with Gly being the only AA not attacked. The two subspecies of Selenomonas tested, differed in their AA degradation patterns. Subspecies lactilytica degraded all AA tested except His and Tyr. Subspecies ruminantium Could not degrade Gly, Leu, lie, Thr, His, Arg, Lys, Trp nor Tyr. Met appeared unique in that it was produced by members of Megaspbaera, Eubacterium and isolate 19D of Streptococcus. Selenomonas, Butyrivibrio and Streptococcus isolated 12D, however, degraded Met. It thus appears that the total ruminal degradation of dietary AA occurs as a result of extensive bacterial interaction. (Key Words: Amino Acid, Degradation, Rumen Bacteria.)
proteolysis. Bacteria involved have been isolated, identified and the proteolytic enzymes studied (Warner, 1956; Hunt and Moore, 1958; Blackburn and Hobson, 1960a,b; Fulghum and Moore, 1963; Borchers, 1965). The fate of free amino acids (AA) has been studied only in experiments designed primarily for the study of proteolysis. In many of these AA catabolism studies, work was performed using high substrate (AA) concentrations and washed or fractionated cell suspensions. Deamination was measured as a function of NH3 production, thus not accounting for those AA utilized by bacterial uptake, binding or decarboxylation. The investigation of AA catabolism involved isolating pure cultures of rumen bacteria that have the ability to use AA for energy and then using these isolates as tools to study AA degradation. Disappearance of 14 AA was measured as a function of microbial growth to obtain results to refute or support a general type deaminase system shared by all bacteria. MATERIALS AND METHODS
Isolation. Samples of rumen fluid were obtained from a 500 kg fistulated steer fed 2 x/day a 16% protein diet consisting of 10 kg alfalfa hay (N.R.C. 1-00-022) and 1 kg Agway Campion Concentrate4 ; water was available ad INTRODUCTION libitum. The animal was maintained on this diet In recent years, much investigative effort has for several months prior to the study. The been directed towards understanding rumen animal was fed at 0800 and 1500 hr and was sampled 2 hr post 0800 feeding. Samples were 1Present address: Eli Lilly and Co., Greenfield strained through one layer of cheesecloth into a Labs., Greenfield, IN 46140. 2,000 ml Erlenmeyer flask warmed to 39 C and 2Present address: Dept. of Dairy Science, Univer- flushed with O2-free CO2 obtained by passing sity of Maryland, College Park, 20740. CO2 through heated copper filings to remove 3Applebrook Research Center, SmithKline Animal any 02. Health Products, 1600 Paoli Pike, West Chester, PA Dilutions were made in the anaerobic dilu19380. 4Agway Inc., Syracuse, NY. Grain products, plant tion fluid of Bryant (1961) and the anaerobic protein products, grain by-products, calcium propioculture techniques of Hungate (1966) and hate, cane molasses, limestone, nono-calcium phosBryant (1972) were employed throughout the phates, dicalcium phosphate, salt, cobalt sulfate, copper sulfate, zinc sulfate, magnesium oxide, vita- study. mins A and E. Bacteria were isolated from roll tubes con821 JOURNAL OF ANIMAL SCIENCE, Vol. 43, No. 4 (1976)
Downloaded from https://academic.oup.com/jas/article-abstract/43/4/821/4697580 by Iowa State University user on 17 January 2019
SrnitbKline Corp. 3, West Cbester, Pennsylvania 19380
822
SCHEIFINGER, RUSSELL AND CHALUPA
s Technicon TSM Amino Acid Analyzer, Technicon Corporation, Tarrytown, NY.
KH2PO4, .6% (NH4)2 SO4, 1.2% NaCI, .24% MgSO4 " 7 H20, .16% CaCI2 9 2 H20) and .1 ml of .1% reaszurin. The following L-amino acids were added to yield a final concentration of 15 mM each: Ala, Arg 9 HC1, Asp, Glu, Gly, His 9 HC1, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp and Val. Tyrosine was added at 10 millimoles. The pH was adjusted to 6.7 and the medium was autoclaved for 15 minutes at 15 lb/in 2 under CO2 in a sealed flask. After cooling to 50 C, 5 ml of sterile 8% Na2 COa and 2 ml of sterile 2.5% Cysteine 9 HC1 were added. The agar medium was then dispensed into 18 • 150 mm tubes stoppered with butyl rubber stoppers under O2-free CO2 in 10 ml amounts. The medium for the initial deaminase test was basically the isolation medium devoid of agar, Cys 9 HC1, Pro, Ser, Trp, Glu, Gly, Ala and Asp. Glucose, cellobiose, maltose and soluble starch were added at .1%, respectively, to provide energy for growth. The amino acid analysis medium was that of the isolation medium except agar and Cys " HCI were deleted, the AA concentration was 1.5 mM and glucose, cellobiose, maltose and soluble starch were added at .1%, respectively, to provide energy. R ESU LTS
Bacteria from the initial isolation were tested for their ability to deaminate a mixture of 10 AA that are considered to be essential for growth and maintenance at the ruminant tissue level, Downes (1961). Organisms giving ~20% deamination based on the decrease in total c~ amino-N were saved and catagorized into genera on the basis of their gross morphological characteristics as shown in table 1. The Selenomonads were further classified into subspecies based on their abilities to grow on lactate and glycerol. The fermentation characteristics of the other isolates supplemented the gross morphological classification into genera. The organisms isolated represent genera to be active in rumen proteolysis (Bladen et al. 1961). Table 2 shows the results of the initial deaminase test. S. ruminantium subsp, lactilytica gave the greatest decrease in c~ amino-N while subsp, ruminantium exhibited the least degradation. The remaining genera gave approximately similar degradation rates somewhere between the two extremes. The results of this test only suggest an
Downloaded from https://academic.oup.com/jas/article-abstract/43/4/821/4697580 by Iowa State University user on 17 January 2019
taining the agar media of Bryant and Burkey (1953) modified to contain AA instead of carbohydrates as energy sources. All incubations were at 39 C and those colonies developing larger than background pin point were picked and maintained on RGCA slants as described by Bryant and Robinson (1961). The isolates were tentatively classified into genera on the basis of their morphology. Carbohydrate fermentation characteristics were also determined and used primarily to distinguish the lactate-using and nonlactate-using species of Setenomonas. Culture Growth f o r Amino Acid and Initial Deamination Analysis. The bacteria were grown in 18 • 150 mm tubes containing 10 ml of the appropriate broth under an atmosphere of 02-free CO2. The medium was inoculated from a 24-hr culture using identical broth and growth was allowed to proceed until an optical density 600 nm of approximately .9 was reached (18 hr.). a Amino N Analysis. Two ml of the fermentation broth was centrifuged at 18,000 x g for 30 minutes. To .25 ml of the cell-free supernatant was added 4.5 ml of .5% TCA and 5 ml washed CuPO4 (Borchers, 1959). The ingredients were mixed, cooled for 30 min at 4 C and then centrifuged at 3,000 x g for 10 minutes. This supernatant was used in the a amino-N Cuprizone assay of Borchers (1959) adapted for automated analysis. Results were determined as the decrease in total a amino-N and expressed as the percentage decrease in t~ amino-N from the amount present in the medium before inoculation. Amino Acid Analysis. Fermentation broth was centrifuged at 18,000 x g for 30 minutes. The resulting supernatant was deproteinized with sulfosalicyclic acid and assayed for amino acids, using norleucine as an internal standard with a Technicon amino acid analyzer s. Results are expressed as percentage decrease in the individual AA compared to the amount present in the medium before inoculation. Medium. Isolation media was prepared by first adding the following ingredients and distilled H 2 0 to a final vol of 93 mh .5 g yeast extract, .1 g dithiothreitol, 1.5% agar, 20 ml clarified rumen fluid (Bryant, 1961), 4 ml each of mineral solution 1 (.6% K2 HPO4 ) and 2 (.6%
AMINO ACID DEGRADATION BY PURE CULTURES OF RUMEN BACTERIA
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