45.2.07 AOAC Of fi cial Method 960.47 - AOAC International

45.2.07
AOAC Of fi cial Method 960.47
Amino Acids
in Vi ta min Prep a ra tions
Mi cro bi o log i cal Methods
First Ac tion 1960
(Ap pli ca ble only to ma te ri als con tain ing free forms of amino
ac ids in ab sence of ap pre cia ble amount of pro tein.)
A. Stock So lu tions for Basal Me dia
(Store all so lu tions un der to lu ene; in ad di tion, store vi ta min and
amino acid so lu tions in dark at ca 10�C.)
(a) Amino acid so lu tion.—Pre pare stock so lu tion of each amino
acid as in Ta ble 960.47A. Use amino ac ids of high est pu rity
avail able. (DL form may be pre ferred since it is less likely to be
con tam i nated with other amino ac ids.) If DL form is used, dou ble
weight given in ta ble. Dis solve amino acid with heat, if nec es sary,
and di lute to in di cated vol ume with H 2O. Where spe cial sol vents are
in di cated, dis solve amino acid in vol ume ini tial sol vent spec i fied,
and di lute to fi nal vol ume with H 2O.
(b) Ad e nine–gua nine–ura cil so lu tion.—See 960.46A(b) (see
45.2.01).
(c) Salt so lu tion A.—Dis solve 50 g an hy drous K 2HPO 4 and 50 g
an hy drous KH 2PO 4 in H 2O, di lute to 500 mL, and add 5 drops HCl.
(d) Salt so lu tion B.—Dis solve 20 g MgSO 4�7H 2O, 1 g NaCl, 1 g
FeSO 4�7H 2O, and 1 g MnSO 4�H 2O in H 2O, di lute to 500 mL, and add
5 drops HCl.
(e) Vi ta min so lu tion I.—Dis solve 25 mg ri bo fla vin, 25 mg
thi a mine�HCl, 50 mg ni a cin, and 0.15 mg bi o tin in 0.02M
CH 3COOH to make 1 L.
(f) Vi ta min so lu tion II.—Dis solve 50 mg pyridoxal, 50 mg
pyridoxamine�HCl, 25 mg cal cium pantothenate, 5 mg
Table 960.47A. Amino acid stock so lu tions
p-ami no ben zoic acid, and 0.25 mg fo lic acid in 25% al co hol to make
1 L.
B. Basal Me dium
Com plete as say me dium is given in Ta ble 960.47B. In pre par ing
me dium for given as say, omit par tic u lar amino acid to be as sayed
from me dium. Con cen tra tion of all in gre di ents in basal me dium is
twice what it will be in as say tube af ter ad di tion of test or stan dard
so lu tion and H 2O. Ta ble lists amounts of in gre di ents re quired for 50,
100, 150, and 200 tubes. Pre pare fresh for each as say.
To com bined so lu tions add glu cose and CH 3COONa with mix ing.
When so lu tion is com plete, ad just to pH 6.8 and di lute to vol ume.
(Note: Amounts of glu cose and CH 3COONa de pend upon or gan ism
used in as say.)
C. Cul ture and Sus pen sion Me dia
(a) Agar cul ture me dium.—See 960.46B(b) (see 45.2.01).
(Avail able from Difco Lab o ra tories as Lactobacilli Agar AOAC,
Cat. No. 290010.)
(b) Liq uid cul ture me dium.—Di lute mea sured vol ume of
com plete amino acid basal me dium, B, with equal vol ume H 2O. Add
10 mL por tions to test tubes, cover to pre vent con tam i na tion (or
screw-cap tubes may be used), ster il ize 15 min in au to clave at
121�–123°C, and cool tubes as rap idly as prac ti ca ble to keep color
for ma tion at min i mum. Store in dark at ca 10°C.
(c) Liq uid sus pen sion me dium.—Di lute mea sured vol ume
ap pro pri ate basal me dium (with out amino acid be ing as sayed), B,
with equal vol ume H 2O. Add 10 mL por tions to test tubes, cover to
pre vent con tam i na tion, ster il ize 15 min at 121�–123�C, and cool as
rap idly as prac ti ca ble to keep color for ma tion at min i mum. Store in
dark at ca 10�C.
Amino acid CAS Registry No. Weight, g Ini tial solvent, mL Fi nal volume, mL
Fi nal concentration,
mg/mL
(a) L-Alanine 56-41-7 5.000 900 H2O 1000 5
(b) L-Arginine�HCl 7048-02-4 12.095 400 H 2 O 500 20
(c) L-Asparagine 70-47-3 5.000 400 H 2 O 500 10
(d) L-As par tic acid 56-84-8 10.000 100 1M NaOH 1000 10
(e) L-Cysteine�HCl 52-89-1 5.000 100 2M HCl 1000 5
(f) L-Cystine 56-89-3 5.000 100 2M HCl 1000 5
(g) L-Glutamic acid 56-86-0 10.000 100 1M NaOH 1000 10
(h) Glycine 56-40-6 10.000 400 H 2 O 500 20
(i) L-Histidine�HCl 7048-02-4 12.351 400 H 2 O 500 20
(j) L-Hydroxyproline 51-35-4 5.000 150 H 2 O 250 20
(k) L-Isoleucine 73-32-5 5.000 900 H 2 O 1000 5
(l) L-Leucine 61-90-5 10.000 900 H 2 O 1000 10
(m) L-Lysine�HCl 657-27-2 6.248 400 H 2 O 500 10
(n) L-Methionine 63-68-3 5.000 900 H 2 O 1000 5
(o) L-Norleucine 327-57-1 5.000 100 2M HCl 1000 5
(p) L-Phenylalanine 63-91-2 5.000 900 H 2 O 1000 5
(q) L-Proline 147-85-3 5.000 150 H 2 O 250 20
(r) L-Serine 56-45-1 5.000 900 H 2 O 1000 5
(s) L-Threonine 72-19-5 5.000 900 H 2 O 1000 5
(t) L-Tryptophan 73-22-3 5.000 50 1M NaOH 500 10
(u) L-Ty ro sine 60-18-4 5.000 50 1M NaOH 500 10
(v) L-Valine 72-18-4 5.000 900 H 2 O 1000 5
2006 AOAC INTERNATIONAL

Table 960.47B. Com po si tion of basal me dium
In gre di ents (stock so lu tions, A) 250 mL, mL 500 mL, mL 750 mL, mL 1 L, mL
(a) Amino acid so lu tions
L-Alanine 10 20 30 40
L-Arginine�HCl 5 10 15 20
L-Asparagine 5 10 15 20
L-As par tic acid 5 10 15 20
L-Cysteine�HCl 5 10 15 20
L-Cystine 5 10 15 20
L-Glutamic acid a
25 50 75 100
Glycine 5 10 15 20
L-Histidine�HCl 5 10 15 20
L-Hydroxyproline 2.5 5 7.5 10
L-Isoleucine 10 20 30 40
L-Leucine 5 10 15 20
L-Lysine�HCl 5 10 15 20
L-Methionine 10 20 30 40
L-Norleucine 10 20 30 40
L-Phenylalanine 10 20 30 40
L-Proline 2.5 5 7.5 10
L-Serine 10 20 30 40
L-Threonine 10 20 30 40
L-Tryptophan 5 10 15 20
L-Ty ro sine 10 20 30 40
L-Valine 10 20 30 40
(b) Adenine–guanine–uracil 5 10 15 20
(c) Salt So lu tion A 2.5 5.0 7.5 10
(d) Salt So lu tion B 2.5 5.0 7.5 10
(e) Vi ta min So lu tion I 10 20 30 40
(f) Vi ta min So lu tion II 10 20 30 40
Glu cose, an hy drous
with S. faecalis 5 g 10 g 15 g 20 g
with L. plantarum or P. cerevisiae 10 g 20 g 30 g 40 g
NaOAc, an hy drous
with S. faecalis 3 g 6 g 9 g 12 g
with L. plantarum or P. cerevisiae 6 g 12 g 18 g 24 g
a For methionine as say use DL-glutamic acid since L-glutamic acid of ten con tains methionine.
D. Stock Cul tures of Test Or gan isms
Pre pare and test cul tures as in 960.46C (see 45.2.01). Use
fol low ing or gan isms:
(a) Strep to coc cus faecalis (faecium).—ATCC No. 9790. For use
in as say of isoleucine, leucine, threonine, tryptophan, valine,
arginine, and histidine.
(b) Lactobacillus plantarum.—ATCC No. 8014. For use in as say of
isoleucine, leucine, methionine, phenylalanine, tryptophan, and valine.
(c) Pediococcus cerevisiae (acidilactici).—ATCC No. 8042. For
use in as say of lysine, methionine, phenylalanine, ty ro sine, cystine,
and histidine.
E. Inoculum
Make trans fer of cells from fresh stock cul ture of ap pro pri ate
or gan ism to ster ile tube con tain ing 10 mL liq uid cul ture me dium,
C(b). In cu bate 16–24 h at 34� � 0.5�C. Un der asep tic con di tions,
cen tri fuge and de cant supernate. Sus pend cells in 10 mL ap pro pri ate
ster ile sus pen sion me dium, C(c).
F. Ref er ence Stan dard So lu tions of Amino Acids
Dry stan dard ized amino ac ids (avail able from ICN
Phar ma ceu ti cals, Inc., Life Sci ences Group) to con stant weight over
P 2O 5 and store in des ic ca tor over P 2O 5. Pre pare stock so lu tion of each
amino acid by ac cu rately weigh ing amount des ig nated in
Ta ble 960.47C in closed sys tem and di lut ing to fi nal vol ume with
H 2O. Pre pare stan dard work ing so lu tion by di lut ing aliquot stan dard
stock so lu tion to des ig nated vol ume with H 2O. Store stan dard stock
so lu tions un der to lu ene in dark at ca 10�C. Pre pare stan dard work ing
so lu tions fresh for each as say. (Do not shake stan dard so lu tions stored
un der to lu ene.)
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Table 960.47C. Prep a ra tion of ref er ence stock and stan dard amino acid so lu tions
Amino acid
G. Cal i bra tion of Pho tom e ter
Using inoculum, E, and stan dard stock so lu tions, Ta ble 960.47C,
pro ceed as in 960.46F (see 45.2.01), in cu bat ing 16–24 h at
34� � 0.5�C. In mea sure ment, use basal me dium, B, for di lut ing.
H. Prep a ra tion of Test Sam ple
Weight,
mg
(a) Liq uids con tain ing free amino ac ids.—Di lute with H 2O to
con cen tra tion re quired to ob tain test solution.
(b) Dry or semidry ma te ri als.—Add H 2O equal in mL to �10 times
dry weight in g of test por tion. Heat 10 min in au to clave at 121�–123�C.
Cool, di lute to vol ume, and fil ter if nec es sary to ob tain test solution.
Stan dard stock
so lu tion fi nal
vol ume, mL
I. As say
Con cen tra tion,
�g/mL
Pro ceed as in 960.46D (see 45.2.01) (dis re gard ref er ence to
titrimetric method), us ing work ing stan dard so lu tions,
Ta ble 960.47C, test so lu tion, H, and basal me dium, Ta ble 960.47B
(omit ting amino acid be ing as sayed). In cu bate 16–24 h at
34� � 0.5�C.
J. De ter mi na tion
Pro ceed as in 960.46G (see 45.2.01).
K. Cal cu la tions
Aliquot,
mL
Pro ceed as in 960.46H (see 45.2.01).
Stan dard work ing
so lu tion fi nal
vol ume, mL
Ref er ences: JAOAC 41, 420, 679(1958); 43, 34(1960).
Con cen tra tion,
�g/mL
L-Arginine�HCl 121 500 200 5 200 5
L-Cystine (dis solve in 100 mL 1M HCl) 100 1000 100 15 500 3
L-Histidine�HCl�H 2 O (CAS-5934-29-2) 135 1000 100 15 500 3
L-Isoleucine 100 1000 100 5 100 5
L-Leucine 100 1000 100
(with L. plantarum) 5 100 5
(with S. faecalis) 10 100 10
L-Lysine�HCl 251 1000 200 15 200 15
L-Methionine 50 1000 50 10 250 2
L-Phenylalanine 100 1000 100 5 100 5
L-Threonine 100 1000 100 5 100 5
L-Tryptophan (dis solve in 100 mL 0.1M NaOH) 40 1000 40 5 250 0.8
L-Ty ro sine (dis solve in 100 mL 0.1M NaOH) 100 1000 100
(with L. plantarum) 5 250 2
(with P. cerevisiae) 10 250 4
L-Valine 100 1000 100
(with L. plantarum) 10 200 5
(with S. faecalis) 10 100 10
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