Amino acids Ing. Petrová Jaroslava Workshop on Official Controls of Feed AGR 46230, 6. 7. 12. 2011, Ankara. Turkey 6.12.2011 ÚKZÚZ - NRL RO Praha 1
Content of this presentation 1. Function of amino acids in nutrition 2. Structure 3. Methods of determination 4. Free amino acids 5. Total content of amino acids 6. Tryptophan 6.12.2011 ÚKZÚZ - NRL RO Praha 2
1. Function of aminoacids in nutrition -Amino acids are building stones of proteins. -20 proteinogenetic AA -Balance of individual AA Optimum animal performance (feed intake, weight gain) is achieved when the protein fed contains an ideal amount and proportion of all essential amino acids. 6.12.2011 ÚKZÚZ - NRL RO Praha 3
1. Function of amino acids in nutrition - Important amino acids Lysine is the most important amino acid for monogastric species, as it is the first limiting amino acid for pigs and the second limiting amino acid for poultry. Methionine is the third limiting amino acid in diets for pigs and the first limiting amino acid in diets for poultry and crucial nutrient to dairy cows.. Threonine is the second limiting amino acid in feed for pigs and the third limiting amino acid in feed for poultry and it is the necessary complement of Lysine. Tryptophan is particularly suitable for young pigs, because it helps to improve feed intake, growth rate and feed efficiency. 6.12.2011 ÚKZÚZ - NRL RO Praha 4
1. Function of aminoacids in nutrition - Important amino acids Histidine is an essential amino acid for growing animals with antioxidant abilities (authorised for salmonids) Isoleucine is an essential amino acid and help to better balance the crude protein Arginine is an essential amino acid for growing animals Guanidinoacetic acid -a precursor of creatine, which is a conditionally essential nutrient for fast-growing animals fed exclusively vegetarian diets. Valine in addition to Lysine, Threonine and Tryptophan reduce nitrogen excreted by animals. -Good balance of aminoacids helps in better utilization of feed. 6.12.2011 ÚKZÚZ - NRL RO Praha 5
2. Structure Amino acids are molecules containing an amine group, a carboxylic acid group 6.12.2011 ÚKZÚZ - NRL RO Praha 6
3. Methods of determination REGULATION (EC) No 152/2009, Annex III, F. Determination of amino acids (CSN) EN ISO 13903 -Animal feeding stuffs - Determination of amino acids content (CSN) EN ISO 13904 -Animal feeding stuffs - Determination of tryptophan content 6.12.2011 ÚKZÚZ - NRL RO Praha 7
Principle 3. Determination of amino acids content - determination of free (synthetic and natural) and total (peptide bound and free) amino acids in feedingstuffs, using an amino acid analyser or HPLC equipment. The method does not distinguish between the salts of amino acids and it cannot differentiate between D and L forms of amino acids. It is not valid for the determination of tryptophan or hydroxy analogues of amino acids 6.12.2011 ÚKZÚZ - NRL RO Praha 8
3. Determination of amino acids content List of aminoacids determined by the method: sum of cystine and cysteine; methionine; lysine; threonine; alanine; arginine; aspartic acid; glutamic acid; glycine; histidine; isoleucine; leucine; phenylalanine; proline; serine; tyrosine; valine 6.12.2011 ÚKZÚZ - NRL RO Praha 9
3. Determination of amino acids content -Extraction with 0,1 mol HCl/l (free amino acids) -Hydrolysis (total amino acids) with (cys, met) or without oxidation -Separation - ion exchange (strong catex) separation method, mobile phase buffers with growing ph value and ion force. -Detection post column derivatization (ninhydrine, OPA - HPLC), pre-column derivatization (dansylchloride) 6.12.2011 ÚKZÚZ - NRL RO Praha 10
4. Free amino acids The free amino acids are extracted with diluted hydrochloric acid. Coextracted nitrogenous macromolecules are precipitated with sulfosalicylic acid and removed by filtration. The filtered solution is adjusted to ph 2,20. The amino acids are separated by ion exchange chromatography and determined by reaction with ninhydrin with photometric detection at 570 nm. 6.12.2011 ÚKZÚZ - NRL RO Praha 11
5. Total amino acids The procedure chosen depends on the amino. Cyst(e)ine and methionine must be oxidised to cysteic acid and methionine sulphone respectively prior to hydrolysis. Tyrosine must be determined in hydrolysates of unoxidized samples. All the other amino acids can be determined in either the oxidised or unoxidised sample. Oxidation is performed at 0 C with a performic acid/phenol mixture. The oxidised or unoxidised sample is hydrolysed with 6 mol/l hydrocholride acid for 23 h. The hydrolysate is adjusted to ph 2,20. The amino acids are separated by ion exchange chromatography and determined by reaction with ninhydrin using photometric detection at 570 nm (440 nm for proline). 6.12.2011 ÚKZÚZ - NRL RO Praha 12
6. Tryptophane For many organisms (including humans), tryptophan is an essential amino acid. This means that it cannot be synthesized by the organism and therefore must be part of its diet. Amino acids, including tryptophan, act as building blocks in protein biosynthesis. In addition, tryptophan functions as a biochemical precursor for Serotonin (a neurotransmitter) and Niacin 6.12.2011 ÚKZÚZ - NRL RO Praha 13
6. Tryptophane REGULATION (EC) No 152/2009, Annex III, F. Determination of amino acids G. Determination of tryptophane (CSN) EN ISO 13904 -Animal feeding stuffs - Determination of tryptophan content 6.12.2011 ÚKZÚZ - NRL RO Praha 14
6. Determination of tryptophane content For the determination of the total tryptophan, the sample is hydrolyzed under alkaline conditions with saturated barium hydroxide solution and heated to 110 C for 20 h. After hydrolysis internal standard is added. For the determination of free tryptophan, the sample is extracted under mild acidic conditions in the presence of internal standard. The tryptophan and the internal standard in the hydrolysate or in the extract are determined by HPLC with fluorescence detection. 6.12.2011 ÚKZÚZ - NRL RO Praha 15
6. Determination of free tryptophane content Sample (1 5 g) is extracted with 100,0 ml 0,1 mol/l hydrochloric acid, internal standard solution is added by shaking. To 10,0 ml of the supernatant solution is added 5 ml 0,5 mol/l ortho-phosphoric acid. ph is adjusted the to 3,0 Add sufficient volume of methanol and dilute with water. Filter a few ml of the solution through a 0,45 μm membrane filter and inject on the HPLC column. Protect standard solution and extracts against direct sunlight. 6.12.2011 ÚKZÚZ - NRL RO Praha 16
6. Determination of total tryptophane content To the sample (0,1 g to 1 g) 8,4 g barium hydroxide octahydrate and 10 ml water is added and mixed mixture is hydrolysed for 30 min to 60 min at 110 (±2) C for 20 h. Add 2,00 ml of internal standard (α-methyl-tryptophan) solution, cool, add 5 ml 0,5 mol/l ortho-phosphoric acid, and neutralize with 6 mol/l HCl to the ph to 3,0 Add sufficient methanol and dilute with water to the defined volume. Filter a few ml of the solution through a 0,45 μm membrane filter before injection on the HPLC column. 6.12.2011 ÚKZÚZ - NRL RO Praha 17
6. Determination of total tryptophane content HPLC determination Chromatographic column (4.2): 125 mm x 4 mm, C 18, 3 μm packing or equivalent Column temperature: Mobile phase : Flow rate: Total run time: Detection wavelength: Injection volume: Room temperature 3,00 g acetic acid + 900 ml water + 50,0 ml solution of 1,1,1-trichloro-2-methyl-2- propanol in methanol (1 g/100 ml). Adjust ph to 5,00 using ethanolamine. Make up to 1000 ml with water. 1 ml/min approximately 34 min excitation: 280 nm, emission: 356 nm 20 μl 6.12.2011 ÚKZÚZ - NRL RO Praha 18
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