Supplementary Material (AMAC-D-11-00106)

Supplementary Material (AMAC-D-11-00106)

Regulatory role for L-arginine in the utilization of amino acids by pig small-intestinal bacteria

Zhao-Lai Dai, Xi-Long Li, Peng-Bin Xi, Jing Zhang, Guoyao Wu*, Wei-Yun Zhu*

Z.-L. Dai, J. Zhang, W.-Y. Zhu, Laboratory of Gastrointestinal Microbiology, Nanjing Agricultural University, Nanjing 210095, China

Z.-L. Dai, X.-L. Li, P.-B. Xi, G. Wu, Department of Animal Science, Texas A&M University, College Station, TX 77843, USA

* Corresponding author: Dr. Wei-Yun Zhu () or Dr. Guoyao Wu ().

Table 1 Effects of L-arginine on the utilization of the arginine-family of AA by pig small-intestinal bacteria1

L-Arginine, mmol/L / Streptococcus sp. / Escherichia coli / Klebsiella sp. / Jejunal bacteria / Ileal bacteria
Percentage of the initial amount, %
L-Arginine
0 / ND2 / ND / ND / ND / ND
0.5 / -17.89 ± 1.50cA / -45.23 ± 1.05aA / -46.47 ± 1.12aA / -30.26 ± 2.82bA / -30.35 ± 3.21bA
1 / -22.32 ± 2.44cB / -30.12 ± 0.53bB / -32.32 ± 0.62aB / -24.16 ± 4.00cB / -17.78 ± 1.29dB
2 / -12.22 ± 1.32dC / -16.92 ± 0.23cC / -22.56 ± 1.14bC / -25.14 ± 0.91aB / -9.92 ± 0.76eC
5 / -6.21 ± 0.63dD / -8.58 ± 0.46cD / -19.41 ± 0.25bD / -25.55 ± 0.35aB / -8.47 ± 0.36cC
L-Citrulline
0 / -34.29 ± 0.97cA / -13.82 ± 3.42dAB / -58.00 ± 1.36bA / -56.09 ± 1.94bA / -64.88 ± 0.76aA
0.5 / -10.09 ± 1.78eBC / -17.16 ± 1.97dA / -43.99 ± 1.63bB / -24.25 ± 1.67cB / -48.74 ± 2.93aB
1 / -14.48 ± 3.34cB / -9.19 ± 0.92cdBC / -38.61 ± 2.86aC / -23.36 ± 1.27bB / -7.39 ± 1.71dC
2 / -14.51 ± 1.86bB / -8.66 ± 2.30cCD / -23.84 ± 1.28aD / -21.27 ± 2.79aB / +16.72 ± 2.67dD
5 / -8.14 ± 0.79bC / -4.40 ± 2.10cD / -7.28 ± 1.74bE / -22.47 ± 3.06aB / +24.58 ± 5.40dE
L-Glutamate
0 / +11.31 ± 0.16dA / -1.22 ± 0.08bB / +23.76 ± 1.35eC / +4.66 ± 0.28cC / -3.47 ± 0.46aA
0.5 / +14.66 ± 0.70dB / -11.54 ± 0.63aA / +13.83 ± 1.28dB / +4.36 ± 0.25cC / -2.10 ± 0.59bA
1 / +14.36 ± 1.31eB / -12.65 ± 0.95aA / +6.54 ± 0.25cA / +3.90 ± 0.59bC / +8.01 ± 0.21dB
2 / +22.32 ± 2.07dC / -12.66 ± 1.22aA / +6.82 ± 0.83cA / -2.95 ± 0.18bB / +9.15 ± 1.24cB
5 / +25.25 ± 0.98eC / -13.91 ± 0.78aA / +6.27 ± 0.53cA / -4.80 ± 0.72bA / +9.67 ± 0.59dB
L-Glutamine
0 / -21.64 ± 0.89cC / -4.45 ± 0.51dB / -22.84 ± 0.42cB / -43.89 ± 0.64b / -43.97 ± 0.79aA
0.5 / -34.47 ± 1.22dB / -42.59 ± 0.26bA / -48.17 ± 0.62aA / -43.05 ± 2.16bc / -40.78 ± 0.45cB
1 / -42.25 ± 3.19bcA / -42.53 ± 0.64bA / -47.79 ± 0.85aA / -41.66 ± 1.32bc / -39.45 ± 1.35cBC
2 / -41.36 ± 1.35cA / -40.95 ± 0.47cA / -46.47 ± 0.73aA / -43.11 ± 2.48b / -34.75 ± 3.20dC
5 / -46.43 ± 0.31aA / -43.19 ± 0.83bA / -46.59 ± 1.09aA / -41.41 ± 1.96b / -35.28 ± 3.54cC
L-Ornithine
0 / +16.73 ± 1.69dA / -10.88 ± 0.98bA / -2.25 ± 0.65cA / -59.66 ± 0.37aA / -60.43 ± 1.49aA
0.5 / +16.65 ± 1.53eA / -11.05 ± 0.62cA / -2.06 ± 0.54dA / -50.39 ± 2.18bB / -56.74 ± 0.76aB
1 / +21.78 ± 2.33eB / +6.88 ± 1.06dB / -3.04 ± 0.90cA / -53.20 ± 1.14aB / -47.26 ± 1.66bC
2 / +24.62 ± 2.19eB / +5.65 ± 0.53dB / -3.40 ± 0.96cA / -53.05 ± 3.13aB / -43.57 ± 3.41bC
5 / +22.41 ± 2.93eB / +7.13 ± 0.92dB / +5.33 ± 0.59cB / -50.43 ± 1.97aB / -44.02 ± 1.78bC
L-Proline
0 / -29.27 ± 1.47aA / -12.11 ± 0.62dC / -18.18 ± 0.81bA / -14.37 ± 0.12cA / -10.41 ± 0.69eA
0.5 / -25.74 ± 0.80aA / -17.27 ± 0.29bA / -16.68 ± 0.91bA / -13.52 ± 0.53cB / -9.72 ± 0.37dA
1 / -16.38 ± 0.56aB / -17.19 ± 0.48aA / -17.79 ± 0.82aA / -13.86 ± 1.42bB / -8.20 ± 0.46cB
2 / -13.69 ± 1.05cB / -16.03 ± 0.17bB / -17.82 ± 0.22aA / -14.17 ± 0.59cAB / -7.47 ± 0.40dB
5 / -14.30 ± 1.02bB / -15.92 ± 0.75aB / -15.67 ± 0.42aB / -11.35 ± 1.52cBC / -7.54 ± 0.54dB

1 Values are means ± SEM, n=4. a-e: Means in a row with superscripts without a common letter differ, P < 0.05; A-E: Means in a column with superscripts without a common letter differ, P < 0.05. Bacteria were incubated in the presence of 0 – 5 mmol/L L-arginine for 3 h. “-” denotes utilization, “+” denotes production.

2 ND, not detectable.

Table 2 Effects of L-arginine on the utilization of the serine- and aspartate-family of AA by pig small-intestinal bacteria1

L-Arginine, mmol/L / Streptococcus sp. / Escherichia coli / Klebsiella sp. / Jejunal bacteria / Ileal bacteria
Percentage of the initial amount, %
L-Alanine
0 / -4.27 ± 0.26dB / -5.12 ± 0.42cB / -3.76 ± 0.34dB / -19.74 ± 0.66bA / -20.57 ± 0.53aA
0.5 / -3.32 ± 0.45eB / -12.67 ± 0.29cA / -8.12 ± 0.77dA / -16.78 ± 1.03bB / -19.72 ± 0.58aA
1 / -7.13 ± 1.06cA / -12.76 ± 0.49bA / -8.19 ± 0.32cA / -16.57 ± 0.99baB / -16.40 ± 1.62aB
2 / -7.92 ± 1.06cA / -12.45 ± 0.48bA / -7.48 ± 0.90cA / -16.03 ± 0.45aB / -13.84 ± 0.73bB
5 / -7.72 ± 0.19dA / -11.81 ± 0.05cA / -7.61 ± 0.48dA / -16.33 ± 0.70aB / -13.41 ± 0.50bB
L-Asparagine
0 / -100.00 ± 0.002a / -100.00 ± 0.002a / -24.39 ± 1.68cC / -45.48 ± 1.07bB / -42.86 ± 1.96bA
0.5 / -100.00 ± 0.002a / -100.00 ± 0.002a / -43.05 ± 1.86bB / -100.00 ± 0.002aA / -44.96 ± 0.52bA
1 / -100.00 ± 0.002a / -100.00 ± 0.002a / -47.96 ± 1.15bA / -100.00 ± 0.002aA / -29.76 ± 3.58cB
2 / -100.00 ± 0.002a / -100.00 ± 0.002a / -48.08 ± 1.57bA / -100.00 ± 0.002aA / -27.15 ± 2.55cB
5 / -100.00 ± 0.002a / -100.00 ± 0.002a / -48.52 ± 1.78bA / -100.00 ± 0.002aA / -31.77 ± 1.82cB
L-Aspartate
0 / -8.94 ± 0.79dB / -100.00 ± 0.002a / -79.91 ± 0.79b / +12.33 ± 1.20eB / -37.66 ± 3.06cA
0.5 / -11.33 ± 0.33cB / -100.00 ± 0.002a / -100.00 ± 0.002a / +14.78 ± 0.92dB / -27.86 ± 0.40bB
1 / -18.84 ± 1.12bA / -100.00 ± 0.002a / -100.00 ± 0.002a / +11.78 ± 0.89cC / -19.06 ± 2.29bC
2 / -19.79 ± 2.82bA / -100.00 ± 0.002a / -100.00 ± 0.002a / +13.95 ± 2.21cB / -18.11 ± 2.58bC
5 / -24.02 ± 1.05bA / -100.00 ± 0.002a / -100.00 ± 0.002a / +15.03 ± 1.91dA / -19.73 ± 0.94cC
Glycine
0 / -2.43 ± 0.48eB / -12.36 ± 0.61cB / -9.83 ± 0.46dC / -21.17 ± 0.78aA / -17.58 ± 0.73bA
0.5 / -3.37 ± 0.65dB / -13.98 ± 0.35bB / -19.77 ± 0.71aB / -19.91 ± 0.77aAB / -8.78 ± 0.39cB
1 / -13.63 ± 0.98cA / -15.25 ± 0.59bA / -22.17 ± 0.97aA / -18.94 ± 0.82bB / -9.19 ± 0.76dB
2 / -14.56 ± 0.46cA / -15.01 ± 0.70cA / -22.56 ± 1.03aA / -17.93 ± 0.74bB / -7.40 ± 0.31dB
5 / -14.13 ± 0.65cA / -15.50 ± 0.64bA / -21.09 ± 0.35aA / -16.34 ± 0.30bC / -8.68 ± 0.42dB
L-Serine
0 / -100.00 ± 0.002a / -100.00 ± 0.002a / -100.00 ± 0.002a / -20.59 ± 0.62cC / -33.53 ± 1.49bA
0.5 / -100.00 ± 0.002a / -100.00 ± 0.002a / -100.00 ± 0.002a / -29.54 ± 0.91cB / -32.46 ± 0.94bA
1 / -100.00 ± 0.002a / -100.00 ± 0.002a / -100.00 ± 0.002a / -26.39 ± 0.51bB / -12.61 ± 1.24cB
2 / -100.00 ± 0.002a / -100.00 ± 0.002a / -100.00 ± 0.002a / -30.51 ± 0.87bA / -11.08 ± 0.24cB
5 / -100.00 ± 0.002a / -100.00 ± 0.002a / -100.00 ± 0.002a / -33.58 ± 0.77bA / -11.52 ± 0.59cB
L-Threonine
0 / -9.63 ± 0.91dC / -42.06 ± 2.03aB / -16.96 ± 0.61cC / -31.73 ± 0.75bA / -39.70 ± 1.83bA
0.5 / -28.52 ± 2.83dB / -48.16 ± 0.73aA / -41.45 ± 1.04bA / -25.26 ± 0.67dB / -35.95 ± 0.36cA
1 / -49.18 ± 7.31aA / -42.75 ± 2.25bB / -43.81 ± 0.54bA / -24.81 ± 1.90cB / -17.28 ± 2.55dB
2 / -52.74 ± 4.49aA / -32.75 ± 1.60cC / -42.60 ± 0.84bA / -26.03 ± 1.98dB / -18.30 ± 0.38eB
5 / -52.44 ± 6.68aA / -29.10 ± 4.15cC / -39.60 ± 0.75bB / -27.39 ± 0.92cB / -15.17 ± 0.96dB

1 Values are means ± SEM, n=4. a-e: Means in a row with superscripts without a common letter differ, P < 0.05; A-D: Means in a column with superscripts without a common letter differ, P < 0.05. Bacteria were incubated in the presence of 0 – 5 mmol/L L-arginine for 3 h. “-” denotes utilization, “+” denotes production.

2 Complete utilization of the corresponding amino acid was observed after 3 h of incubation.

Table 3 Effects of L-arginine on the utilization of branched-chain AA by pig small-intestinal bacteria1

L-Arginine, mmol/L / Streptococcus sp. / Escherichia coli / Klebsiella sp. / Jejunal bacteria / Ileal bacteria
Percentage of the initial amount, %
L-Isoleucine
0 / -1.61 ± 0.22eC / -11.71 ± 0.20cA / -7.35 ± 1.12dB / -20.83 ± 1.31bA / -32.20 ± 1.64aA
0.5 / -5.36 ± 0.45dB / -10.34 ± 1.08cAB / -10.00 ± 0.89cAB / -14.69 ± 1.91bB / -32.30 ± 0.53aA
1 / -8.06 ± 1.13cA / -8.52 ± 0.29cBC / -14.72 ± 2.23aA / -13.96 ± 1.10bB / -16.30 ± 2.56aB
2 / -9.09 ± 0.86cA / -7.95 ± 0.98cC / -13.61 ± 0.66bA / -16.03 ± 1.44aB / -18.09 ± 1.19aB
5 / -8.87 ± 0.32cA / -4.47 ± 1.23cD / -12.98 ± 0.43bA / -16.87 ± 0.97aB / -17.59 ± 1.15aB
L-Leucine
0 / -1.05 ± 0.49eB / -7.75 ± 0.62cA / -3.58 ± 1.07dB / -17.61 ± 1.45bA / -27.31 ± 0.82aA
0.5 / -3.36 ± 0.94dA / -8.58 ± 0.52cA / -5.90 ± 0.93cB / -12.30 ± 1.68bB / -22.94 ± 1.35aB
1 / -3.22 ± 0.55dA / -5.98 ± 0.30cB / -13.61 ± 1.09aA / -11.86 ± 0.72bB / -14.18 ± 0.69aC
2 / -3.82 ± 0.50cA / -6.17 ± 0.25bB / -12.60 ± 0.80aA / -12.31 ± 0.97aB / -13.77 ± 0.86aC
5 / -3.47 ± 0.25dA / -6.43 ± 0.26cB / -13.61 ± 0.42aA / -13.80 ± 1.64aB / -11.63 ± 0.86bC
L-Valine
0 / -5.78 ± 0.87eB / -13.30 ± 1.26cC / -8.29 ± 0.69dC / -19.46 ± 1.43b / -28.24 ± 0.95aA
0.5 / -7.69 ± 0.94eB / -16.43 ± 0.79bA / -11.31 ± 0.90dB / -15.88 ± 0.63c / -25.59 ± 1.07aA
1 / -12.56 ± 1.30bA / -16.36 ± 2.58aA / -18.43 ± 2.37aA / -14.38 ± 2.11a / -14.45 ± 2.32aB
2 / -12.81 ± 2.03abA / -10.32 ± 2.36bB / -17.78 ± 0.61aA / -16.69 ± 1.74a / -13.42 ± 2.28bB
5 / -12.38 ± 0.52cA / -10.19 ± 1.49dB / -17.45 ± 0.42aA / -16.83 ± 1.67ab / -15.25 ± 1.41bB

Table 4 Effects of L-arginine on the utilization of sulfur AA by pig small-intestinal bacteria1

L-Arginine, mmol/L / Streptococcus sp. / Escherichia coli / Klebsiella sp. / Jejunal bacteria / Ileal bacteria
Percentage of the initial amount, %
L-Cystine
0 / -7.80 ± 0.82cA / -22.88 ± 1.19aA / -14.33 ± 0.60bA / -7.63 ± 1.25c / -14.74 ± 1.10bA
0.5 / -5.58 ± 1.85dAB / -23.90 ± 1.31aA / -12.77 ± 0.44bA / -6.50 ± 1.50d / -10.14 ± 1.65cB
1 / -2.02 ± 0.50dB / -23.64 ± 1.34aA / -13.15 ± 0.53bA / -9.29 ± 0.90c / -10.41 ± 1.86cB
2 / -3.48 ± 0.75dB / -21.11 ± 1.21aAB / -4.49 ± 0.31dB / -7.24 ± 1.17c / -10.71 ± 1.76bB
5 / -2.32 ± 0.80dB / -19.26 ± 1.11aB / -5.10 ± 0.72cB / -7.66 ± 1.67b / -7.59 ± 0.92bB
L-Methionine
0 / -6.92 ± 1.42dB / -19.91 ± 0.70cA / -8.28 ± 1.24dC / -23.46 ± 1.15b / -29.58 ± 0.84aA
0.5 / -7.45 ± 1.59dB / -25.47 ± 0.93bA / -20.00 ± 0.84cB / -21.73 ± 1.65c / -30.71 ± 0.93aA
1 / -16.48 ± 1.23cA / -24.22 ± 1.13bA / -30.04 ± 0.71aA / -18.57 ± 1.98c / -17.63 ± 1.59cB
2 / -16.32 ± 2.38bA / -16.87 ± 0.61bC / -22.95 ± 0.85aB / -20.91 ± 1.52a / -17.08 ± 1.42bB
5 / -12.97 ± 0.99cA / -15.81 ± 1.73bC / -22.56 ± 0.81aB / -19.64 ± 2.45b / -18.16 ± 0.69bB

1 Values are means ± SEM, n=4. a-d: Means in a row with superscripts without a common letter differ, P < 0.05; A-D: Means in a column with superscripts without a common letter differ, P < 0.05. Bacteria were incubated in the presence of 0 – 5 mmol/L L-arginine for 3 h. “-” denotes utilization.

Table 5 Effects of L-arginine on the utilization of aromatic AA by pig small-intestinal bacteria1

L-Arginine, mmol/L / Streptococcus sp. / Escherichia coli / Klebsiella sp. / Jejunal bacteria / Ileal bacteria
Percentage of the initial amount, %
L-Phenylalanine
0 / -0.46 ± 0.76dB / -5.25 ± 0.66cB / -4.87 ± 0.48cB / -13.98 ± 0.40b / -29.74 ± 1.38aA
0.5 / -0.68 ± 0.55dB / -9.05 ± 0.96cA / -10.81 ± 0.86cA / -14.38 ± 1.13b / -31.35 ± 0.39aA
1 / -10.49 ± 1.01bcA / -9.46 ± 0.78cA / -11.03 ± 0.93bA / -14.67 ± 1.59a / -16.92 ± 0.71aB
2 / -10.93 ± 1.07bA / -9.46 ± 0.61bA / -10.92 ± 0.69bA / -13.93 ± 0.89a / -15.28 ± 2.12aB
5 / -10.81 ± 1.40bcA / -8.86 ± 1.64cA / -11.53 ± 0.74bA / -16.13 ± 0.57a / -16.40 ± 1.72aB
L-Tryptophan
0 / -4.52 ± 0.92dB / -16.26 ± 1.21c / -25.86 ± 1.64bA / -7.47 ± 2.49dC / -34.60 ± 4.12a
0.5 / -4.35 ± 0.75cB / -14.93 ± 4.10b / -15.44 ± 0.31bB / -17.66 ± 1.63bB / -33.27 ± 1.54a
1 / -8.09 ± 2.50cA / -13.31 ± 4.64b / -9.25 ± 0.58cC / -15.49 ± 1.57bB / -30.59 ± 1.50a
2 / -8.58 ± 1.23cA / -12.86 ± 5.27bc / -9.77 ± 1.13cC / -15.96 ± 1.44bB / -27.44 ± 1.29a
5 / -7.75 ± 0.96dA / -12.06 ± 4.34c / -6.11 ± 1.55dD / -26.06 ± 2.61bA / -30.52 ± 0.89a
L-Tyrosine
0 / -9.02 ± 0.25cB / -4.41 ± 1.02dB / -3.17 ± 0.48dD / -16.51 ± 2.70bB / -19.35 ± 2.35aA
0.5 / -11.32 ± 0.42dB / -15.43 ± 0.65cA / -6.97 ± 0.94eC / -25.80 ± 4.88aA / -19.25 ± 0.87bA
1 / -21.31 ± 1.38bA / -15.45 ± 0.56cA / -12.70 ± 1.34dB / -26.47 ± 4.34aA / -16.86 ± 1.58cA
2 / -19.67 ± 0.83bA / -16.82 ± 1.87bA / -11.58 ± 0.72cB / -30.77 ± 5.58aA / -11.45 ± 0.55cB
5 / -20.44 ± 1.73bA / -17.13 ± 0.27cA / -29.12 ± 0.55aA / -28.31 ± 3.06aA / -11.42 ± 0.82dB

Table 6 Effects of L-arginine on the utilization of lysine and histidine by pig small-intestinal bacteria1

L-Arginine, mmol/L / Streptococcus sp. / Escherichia coli / Klebsiella sp. / Jejunal bacteria / Ileal bacteria
Percentage of the initial amount, %
L-Histidine
0 / -3.80 ± 0.10cE / -33.18 ± 2.88aA / -27.93 ± 1.80b / -33.59 ± 0.46a / -33.63 ± 0.92aB
0.5 / -9.01 ± 1.86cD / -35.94 ± 1.28aA / -30.93 ± 0.98b / -35.64 ± 1.46a / -39.47 ± 1.67aA
1 / -18.90 ± 0.94dC / -28.68 ± 0.63cB / -31.27 ± 1.53b / -33.76 ± 0.61a / -33.89 ± 1.60aB
2 / -21.27 ± 0.88cB / -28.47 ± 1.17bB / -32.25 ± 3.19a / -33.96 ± 1.02a / -31.48 ± 1.96aC
5 / -33.90 ± 2.56bA / -29.06 ± 1.43dB / -29.96± 0.88d / -35.71 ± 2.26a / -31.35 ± 3.57cdC
L-Lysine
0 / -61.57 ± 0.61bA / -10.49 ± 0.61eC / -71.44 ± 0.55a / -41.40 ± 0.61dA / -48.38 ± 1.50cA
0.5 / -32.19 ± 1.02dB / -14.02 ± 1.24eB / -73.40 ± 1.82a / -35.20 ± 0.45cB / -48.50 ± 1.69bA
1 / -29.57 ± 1.35cB / -13.64 ± 0.44eB / -75.25 ± 0.89a / -34.83 ± 1.46bB / -23.06 ± 2.61dB
2 / -29.99 ± 1.68cB / -21.56 ± 0.38dA / -76.09 ± 1.12a / -35.86 ± 2.49bB / -16.69 ± 1.60eC
5 / -29.87 ± 1.42cB / -22.35 ± 0.84dA / -71.32 ± 2.72a / -36.20 ± 1.63bB / -17.48 ± 1.57eC