ANKOM TDF Dietary - Manual (Page 89)

Operator’s Manual
Rev E 12/2/14
pg. 89
Appendix A – Reagents (AOAC 991.43, AOAC 985.29)
Use Distilled Water throughout.
Solutions common to both AOAC 991.43 and AOAC 985.29 methods
(a) Ethanol 95%.
(b) Ethanol 78%—Place 821 ml of 95% ethanol into a 1 L volumetric flask. Dilute to volume with H
2
O.
(c) Enzyme solutions—To make your enzyme solutions for use in the ANKOM
TDF
Dietary Fiber Analyzer, dilute
ANKOM Concentrate enzymes (TDF80/TDF81, TDF82/TDF83, TDF84/TDF85) with H
2
O according to the Enzyme
Dilution Table below.
Enzyme Dilution Table
Enzyme Dilution
α-Amylase (ANKOM Concentrate) Dilute 5 ml of amylase to 25 ml
Protease (ANKOM Concentrate) Dilute 5 ml of protease to 25 ml
Amyloglucosidase (ANKOM Concentrate) Dilute 5 ml of Amyloglucosidase to 25 ml
The ANKOM TDF Dietary Fiber Analyzer delivers 1 ml of each enzyme solution to
each of the six stations per run (6 ml of each enzyme solution). If you are NOT
using the ANKOM Concentrate enzymes, you must prepare enzyme solutions that
include 0.02% w/v of Sodium Azide (to prevent microbial growth) and the
following enzyme concentrations:
α-Amylase: 150 Ceralpha Units per ml
Protease: 35 Tyrosine Units per ml
Amyloglucosidase: 652 Units per ml
(d) Diatomaceous earth (DE)—(ANKOM XTC, or equivalent).
(e) Acetone—reagent grade.
Solutions unique to the AOAC 991.43 method
(f) MES—2-(N-Morpholino)ethanesulfonic acid (No. M-8250, Sigma Chemical Co., or equivalent).
(g) TRIS—Tris(hydroxymethyl)aminomethane (No. T-1503, Sigma Chemical Co., or equivalent).
(h) MES-TRIS buffer solution—0.05M MES, 0.05M TRIS, pH 8.2 at 24°C. Dissolve 19.52 g MES and 12.2 g TRIS in 1.7
L H
2
O. Adjust pH to 8.2 with 6N NaOH, and dilute to 2 L with H
2
O. (Note: It is important to adjust pH to 8.2 at
24°C. However, if buffer temperature is 20°C, adjust pH to 8.3; if temperature is 28°C, adjust pH to 8.1. For
deviations between 20 and 28°C, adjust by interpolation.)
(i) Hydrochloric acid solution (AOAC 991.43)—0.561N. Dilute 93.5 ml of 6N HCl to 1 L with H
2
O.
Solutions unique to the AOAC 985.29 method
(j) Hydrochloric acid solution (AOAC 985.29)—0.65N. Dilute 325 ml of 1N HCl to 500 ml with H
2
O.
(k) Phosphate buffer solution—0.08M, pH 6.0. Dissolve 1.40 g Sodium Phosphate dibasic, anhydrous (Na
2
HPO
4
) (or 1.75
g dihydrate) and 9.68 g Sodium Phosphate monobasic monohydrate (NaH
2
PO
4
) (or 10.94 g dihydrate) in a 1 L
volumetric flask. Dilute to 1 L with H
2
O. Adjust pH to 6.0.
(l) Sodium Hydroxide Solution—0.55N. Dissolve 11.00 g Sodium Hydroxide (NaOH ACS) in a 500 ml volumetric flask.
Dilute to 500 ml with H
2
O.
To achieve the appropriate volume in the filter bags, the concentrations of the HCl
and NaOH solutions were modified to accomplish the pH adjustment in a manner
equivalent to the AOAC 985.29 method
.
To avoid microbial growth, make all buffer solutions fresh and do NOT allow them
to sit unused for more than two days.