Total Dietary Fiber Video Method (AOAC Method 991.43/AACC method 32-07.01) with K-TDFR

Summary

This video outlines the Lee method (AOAC Method 991.43) for measuring dietary fiber, focusing on the separation of high molecular weight dietary fiber (HMWDF) into insoluble dietary fiber (IDF) and soluble dietary fiber precipitate (SDFP). The method, a modification of the Prosky method, simplifies enzymatic incubations and includes a filtration step for better fiber separation. The tutorial covers the preparation and analysis process, reagent handling, and detailed steps for accurate fiber measurement, including protein and ash analysis. It also highlights the use of MegaCalc for easy dietary fiber calculations, along with tips for sample preparation and method accuracy.

Takeaways

• 😀 The Lee method (AOAC Method 991.43) is used for the separate measurement of high molecular weight dietary fiber (HMWDF), including insoluble dietary fiber (IDF) and soluble dietary fiber precipitate (SDFP).
• 😀 The Lee method is a modified version of the Prosky method, simplifying the enzymatic incubation conditions and introducing a filtration step to separate fiber types.
• 😀 The method involves incubating samples with α-amylase, protease, and amyloglucosidase to break down starches and proteins, mimicking digestion.
• 😀 Insoluble dietary fiber (IDF) is separated from soluble dietary fiber (SDFP) by filtration, with the residues being washed and dried for further analysis.
• 😀 The total dietary fiber (TDF) value is calculated as the sum of IDF and SDFP, with each fiber component analyzed for protein and ash content.
• 😀 The method has been officially validated by AOAC in 1991 and is widely used for fiber analysis due to its simplicity and practicality.
• 😀 Non-digestible oligosaccharides are not measured in the Lee method, and it may yield inaccurate values for resistant starch.
• 😀 For accurate results, the samples should be dried, fat-free, and ground to pass through a 0.5 mm screen before analysis.
• 😀 The MegaCalc spreadsheet is provided for simplifying the calculations of dietary fiber content, and it can automatically calculate fiber percentages once sample data is entered.
• 😀 Enzyme purity plays a crucial role in ensuring accurate dietary fiber measurements, as discussed in related studies and papers.

Q & A

What is the Lee method for dietary fiber measurement?

The Lee method is a modification of the Prosky method (AOAC Method 991.43) used for measuring high molecular weight dietary fiber (HMWDF). It separates the dietary fiber into insoluble dietary fiber (IDF) and soluble dietary fiber precipitate (SDFP) using enzymatic treatments and ethanol precipitation.

What are the key differences between the Lee method and the Prosky method?

The key differences include simplified enzymatic incubation conditions in the Lee method, where both α-amylase and protease incubations occur in the same buffer. Additionally, the Lee method introduces a filtration step before the ethanol precipitation step to separate IDF and SDFP.

How does the Lee method separate soluble and insoluble dietary fiber?

In the Lee method, insoluble dietary fiber (IDF) is filtered and washed with warm distilled water, while soluble dietary fiber (SDFP) is precipitated from the combined solution of filtrate and water washings using 95% ethanol.

Why are non-digestible oligosaccharides not measured in the Lee method?

The Lee method specifically measures high molecular weight polysaccharides, including water-insoluble fibers and those that precipitate in 80% ethanol. Non-digestible oligosaccharides are not included in the measurement as they do not fit the criteria for high molecular weight polysaccharides.

What is the purpose of the filtration step in the Lee method?

The filtration step in the Lee method helps to separate the insoluble dietary fiber (IDF) from the soluble dietary fiber (SDFP), allowing for accurate measurement of both fractions.

What temperature is required during the enzymatic incubations in the Lee method?

During the Lee method, the α-amylase is incubated at 95-98°C for 30 minutes, protease at 60°C for 30 minutes, and amyloglucosidase at 60°C for a further 30 minutes.

What is the significance of the MegaCalc spreadsheet for the Lee method?

The MegaCalc spreadsheet simplifies the dietary fiber calculations in the Lee method. By inputting sample details, residue weights, and protein and ash content, the spreadsheet automatically calculates the total dietary fiber (TDF), including the individual fractions (IDF and SDFP).

What steps are involved in the preparation of samples for the Lee method?

Samples are milled to pass a 0.5 mm screen and dried in a vacuum or forced air oven. If the sample has over 10% fat content, it is defatted using petroleum ether or an ANKOM XT15 Extractor before analysis.

What is the purpose of the ethanol precipitation step in the Lee method?

The ethanol precipitation step helps to separate the soluble dietary fiber (SDFP) from the solution, where the soluble polysaccharides precipitate out upon the addition of 95% ethanol.

Why is it important to analyze both protein and ash content in the Lee method?

Protein and ash content in the fiber residues need to be analyzed and subtracted from the residue weights to ensure accurate calculations of the dietary fiber content, as both protein and ash can contribute to the overall residue weight.