🍎 Dietary fiber — enzymatic-gravimetric
In short
Determination of total, soluble, and insoluble dietary fiber in food — Enzymatic-gravimetric method (AOAC 991. The test is performed according to AOAC Official Method 991.43; Measurement range: 0.5–100% fiber. The procedure comprises 8 steps (about 18 h 55 min in total); it is used for: Fiber declaration on food labels (EU Reg. 1169/2011), Quality control of cereal products (bread, muesli, bran), Analysis of dietetic and functional products.
At a glance
- Standard: AOAC Official Method 991.43
- Category: Food Chemistry
- Procedure steps: 8
- Total time of stages: 18 h 55 min
- Measurement range: 0.5–100% fiber
- Fractions: TDF (total), IDF (insoluble), SDF (soluble)
- Sample size: 1 g (in duplicate)
Overview
Dietary fiber is a food component important for health (digestion regulation, cholesterol reduction, glycemic control). Its content is mandatory on food labels (EU Reg. 1169/2011).
AOAC method 991.43 involves enzymatic hydrolysis of starch (α-amylase, protease, amyloglucosidase), filtration, precipitation of soluble fraction with ethanol, and gravimetric determination of insoluble and soluble fiber after protein and ash correction. Total fiber = insoluble + soluble.
Method principle
Defatted sample is subjected to sequential enzymatic hydrolysis: (1) thermostable α-amylase (95°C, 30 min) — starch hydrolysis, (2) protease (60°C, 30 min) — protein hydrolysis, (3) amyloglucosidase (60°C, 30 min) — dextrin hydrolysis. The residue is filtered — insoluble dietary fiber (IDF) remains on filter. Filtrate is precipitated with 4 volumes of 95% ethanol (60°C, 1 h), filtered — precipitate is soluble dietary fiber (SDF). Both filters are dried, weighed, and corrected for protein (Kjeldahl) and ash (550°C).
Applications
- Fiber declaration on food labels (EU Reg. 1169/2011)
- Quality control of cereal products (bread, muesli, bran)
- Analysis of dietetic and functional products
- Analysis of vegetables, fruits, and their products
- Feed and feed mixture control
- Health claims related to fiber
Key parameters
| Parameter | Value |
|---|---|
| Measurement range | 0.5–100% fiber |
| Fractions | TDF (total), IDF (insoluble), SDF (soluble) |
| Sample size | 1 g (in duplicate) |
| Precision (CV) | 3–8% |
| Analysis time | 2 days (enzymes + drying + Kjeldahl + ash) |
Standard
- Standard number
- AOAC Official Method 991.43
- Title (PL)
- Oznaczanie całkowitego, rozpuszczalnego i nierozpuszczalnego błonnika pokarmowego w żywności — Metoda enzymatyczno-grawimetryczna
- Title (EN)
- Total, Soluble and Insoluble Dietary Fiber in Foods — Enzymatic-Gravimetric Method
Step-by-step procedure
-
Defatting (if >10% fat)
Extract with petroleum ether (3× Soxhlet). Dry residue.
-
α-Amylase hydrolysis
Weigh 1 g sample (duplicate). Add 40 mL MES-TRIS buffer + 50 µL α-amylase. Incubate 95°C / 30 min.
-
Protease hydrolysis
Cool to 60°C. Add 100 µL protease. Incubate 60°C / 30 min with mixing.
-
Amyloglucosidase hydrolysis
Acidify to pH 4.1–4.8 (0.56 mol/L HCl). Add 200 µL amyloglucosidase. Incubate 60°C / 30 min.
-
Filtration (IDF)
Filter through crucible with Celite. Wash with warm water (70°C), 95% ethanol, acetone. Dry 105°C.
-
SDF precipitation
Add 4 vol. 95% EtOH (60°C) to filtrate. Incubate 60 min. Filter through crucible with Celite. Dry 105°C.
-
Protein + ash correction
From one duplicate determine protein (Kjeldahl), from other ash (525°C/5 h). Subtract from residue mass.
-
Calculations
TDF [%] = [(residue − protein − ash − blank) / sample mass] × 100. TDF = IDF + SDF.
Required equipment and apparatus
| Equipment | Example | Indicative price |
|---|---|---|
| Shaking water bath | GFL 1083, Memmert WNB 45 (60–100°C) | 5 000–15 000 PLN |
| Filtering crucibles (fritted) | Gooch crucibles with porous frit + Celite | 30–80 PLN/pc |
| Drying oven 105°C | Binder FD 56 | 5 000–15 000 PLN |
| Muffle furnace 525°C | Nabertherm L 9/11, Carbolite CWF | 10 000–30 000 PLN |
| Kjeldahl system | BÜCHI K-436 + K-360 | 70 000–140 000 PLN |
| Analytical balance 0.1 mg | Mettler Toledo ME204 | 5 000–15 000 PLN |
Reagents, media and consumables
| Reagent | CAS | Details |
|---|---|---|
| Thermostable α-amylase | 9000-90-2 | Megazyme E-BLAAM, activity ≥3000 U/mL, from Bacillus licheniformis |
| Protease | 9001-92-7 | Megazyme E-BSPRT, from Bacillus subtilis |
| Amyloglucosidase | 9032-08-0 | Megazyme E-AMGDF, from Aspergillus niger |
| Ethanol 95% | 64-17-5 | For SDF precipitation (4 vol. to 1 vol. filtrate) |
| MES-TRIS buffer pH 8.2 | — | MES 0.05 mol/L + TRIS 0.05 mol/L |
| Celite (diatomaceous earth) | 68855-54-9 | Filter aid for crucible frit |
Health and safety (OHS)
- Water bath 95°C — burn risk, thermal gloves
- Ethanol 95% — flammable, keep away from fire
- Muffle furnace 525°C — open only after cooling to <200°C
- Acids and bases for pH adjustment — gloves and safety glasses
Frequently asked questions
Which standard describes this test?
The test is performed according to AOAC Official Method 991.43 — “Total, Soluble and Insoluble Dietary Fiber in Foods — Enzymatic-Gravimetric Method”.
How does this method work?
Defatted sample is subjected to sequential enzymatic hydrolysis: (1) thermostable α-amylase (95°C, 30 min) — starch hydrolysis, (2) protease (60°C, 30 min) — protein hydrolysis, (3) amyloglucosidase (60°C, 30 min) — dextrin hydrolysis. The residue is filtered — insoluble dietary fiber (IDF) remains on filter.
What is the measuring range and accuracy?
Measurement range: 0.5–100% fiber; Fractions: TDF (total), IDF (insoluble), SDF (soluble); Sample size: 1 g (in duplicate); Precision (CV): 3–8%.
How long does the test take?
The times given for the individual stages add up to approximately 18 h 55 min. The procedure comprises 8 steps.
What equipment is required?
Shaking water bath, Filtering crucibles (fritted), Drying oven 105°C, Muffle furnace 525°C, Kjeldahl system, Analytical balance 0.1 mg. Indicative cost of the core instrument (Shaking water bath): 5 000–15 000 PLN.
Where is this test used?
Fiber declaration on food labels (EU Reg. 1169/2011); Quality control of cereal products (bread, muesli, bran); Analysis of dietetic and functional products; Analysis of vegetables, fruits, and their products; Feed and feed mixture control; Health claims related to fiber.
What safety precautions apply?
Water bath 95°C — burn risk, thermal gloves; Ethanol 95% — flammable, keep away from fire; Muffle furnace 525°C — open only after cooling to <200°C; Acids and bases for pH adjustment — gloves and safety glasses.
Which laboratory can perform this test?
The test is performed by laboratories accredited to ISO/IEC 17025. On LabCoda you can find them by the standard number AOAC 991.43.