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LabCoda / Knowledge Base / Dietary fiber — enzymatic-gravimetric

🍎 Dietary fiber — enzymatic-gravimetric

Food Chemistry AOAC 991.43

Determination of total, soluble, and insoluble dietary fiber in food — Enzymatic-gravimetric method (AOAC 991.43). Nutritional value parameter.

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

Key parameters

ParameterValue
Measurement range0.5–100% fiber
FractionsTDF (total), IDF (insoluble), SDF (soluble)
Sample size1 g (in duplicate)
Precision (CV)3–8%
Analysis time2 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

1. Defatting (if >10% fat)

Extract with petroleum ether (3× Soxhlet). Dry residue.

⏱ Time: 4 h

2. α-Amylase hydrolysis

Weigh 1 g sample (duplicate). Add 40 mL MES-TRIS buffer + 50 µL α-amylase. Incubate 95°C / 30 min.

⏱ Time: 35 min • 🌡 Temperature: 95°C

3. Protease hydrolysis

Cool to 60°C. Add 100 µL protease. Incubate 60°C / 30 min with mixing.

⏱ Time: 35 min • 🌡 Temperature: 60°C

4. Amyloglucosidase hydrolysis

Acidify to pH 4.1–4.8 (0.56 mol/L HCl). Add 200 µL amyloglucosidase. Incubate 60°C / 30 min.

⏱ Time: 35 min • 🌡 Temperature: 60°C

5. Filtration (IDF)

Filter through crucible with Celite. Wash with warm water (70°C), 95% ethanol, acetone. Dry 105°C.

⏱ Time: 2 h • 🌡 Temperature: 105°C

6. SDF precipitation

Add 4 vol. 95% EtOH (60°C) to filtrate. Incubate 60 min. Filter through crucible with Celite. Dry 105°C.

⏱ Time: 3 h • 🌡 Temperature: 60°C

7. Protein + ash correction

From one duplicate determine protein (Kjeldahl), from other ash (525°C/5 h). Subtract from residue mass.

⏱ Time: 8 h

8. Calculations

TDF [%] = [(residue − protein − ash − blank) / sample mass] × 100. TDF = IDF + SDF.

⏱ Time: 10 min

Required equipment and apparatus

EquipmentExampleIndicative price
Shaking water bathGFL 1083, Memmert WNB 45 (60–100°C)5 000–15 000 PLN
Filtering crucibles (fritted)Gooch crucibles with porous frit + Celite30–80 PLN/pc
Drying oven 105°CBinder FD 565 000–15 000 PLN
Muffle furnace 525°CNabertherm L 9/11, Carbolite CWF10 000–30 000 PLN
Kjeldahl systemBÜCHI K-436 + K-36070 000–140 000 PLN
Analytical balance 0.1 mgMettler Toledo ME2045 000–15 000 PLN

Reagents, media and consumables

ReagentCASDetails
Thermostable α-amylase9000-90-2Megazyme E-BLAAM, activity ≥3000 U/mL, from Bacillus licheniformis
Protease9001-92-7Megazyme E-BSPRT, from Bacillus subtilis
Amyloglucosidase9032-08-0Megazyme E-AMGDF, from Aspergillus niger
Ethanol 95%64-17-5For 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)68855-54-9Filter aid for crucible frit

Health and safety (OHS)

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