AOAC 991.43 Total, Soluble and Insoluble Dietary Fiber in Foods - Enzymatic-Gravimetric Method MES-TRIS Bufler A

According to the “AOAC 991.43 Total, soluble and insoluble dietary fiber in foods - Enzymatic-gravimetric method MES-TRIS Bufler A” standard developed by the Official Association of Analytical Chemists (AOAC), two samples each of dried foods (defatted if they contain more than 10% fat) are subjected to sequential enzymatic digestion with heat-resistant α-amylase, protease, and amyloglucosidase to remove starch and protein. For total dietary fiber, the enzyme digestion product is treated with alcohol to precipitate the soluble dietary fiber before filtration, and the total dietary fiber residue is washed with alcohol and acetone, dried, and weighed. For insoluble and soluble dietary fiber, the enzyme digestion product is filtered, and the residual insoluble dietary fiber is washed with warm water, dried, and weighed. For soluble dietary fiber, the combined filter and residue are precipitated with alcohol, filtered, dried, and weighed. Total dietary fiber, insoluble and soluble dietary fiber residual values ​​are corrected for protein, ash and empty space.

The following equipment is used in the tests:

• Beakers: 400 or 600 mL (in any form).
• Filtering crucible: Sintered disc, coarse-grained, ASTM 40-60 Hm pore size, 60 mL Pyrex. It is prepared as follows: It is incinerated overnight in an ash furnace at 525 degrees. Before removing the crucibles, the furnace temperature is allowed to drop below 130 degrees. The crucibles are then left to soak in a 2% cleaning solution at room temperature for 1 hour.

The crucibles are rinsed with normal water and then with deionized water. 15 mL of acetone is used for the final rinse, and then they are air-dried. Approximately 1,0 g of Celite is added to the dry crucibles and dried at 130 degrees to a constant weight. The crucible is cooled in a desiccant for approximately 1 hour, and the weight of the crucible plus Celite is recorded to the nearest 0,1 mg.

• Vacuum system: Vacuum pump or aspirator with regulating device. Thick-walled 1 L filter bottle with side arms. Rubber ring adapters for use with filtration. Masks.
• Shaking water baths: With an automatic on/off timer, it can maintain a temperature of 98 degrees plus/minus 2 degrees. A constant temperature can be set to 60 degrees.
• Balance: Analytical, accuracy plus/minus 0,1 mg.
• Muffle oven: Can maintain a temperature of 525 degrees plus/minus 5 degrees.
• Oven: Can maintain temperatures between 105 and 130 degrees Celsius plus/minus 3 degrees.
• Desiccant: Silicon dioxide or equivalent desiccant. Every two weeks, the desiccant is dried overnight at 130 degrees.
• pH meter: Temperature compensated, pH values ​​standardized with buffer solutions of 4, 0, 7, 0 and 10, 0.
• Pipettes: With disposable tips. 100-300 µL and 5 mL capacity.
• Distributors.
• Magnetic stirrers and stirring rods.

The AOAC 991.43 standard is one of the most widely recognized and historically significant formal methods for determining dietary fiber in foods. Since its initial publication in 1991, this method has been a cornerstone of dietary fiber analysis. Prior to this standard, dietary fiber was generally measured only as crude fiber. The method described in the AOAC 920.86 standard severely underestimated the true dietary fiber content, capturing only a small fraction of cellulose and lignin while losing most of hemicellulose, pectin, gum, and resistant starch.

The AOAC 991.43 standard was developed to provide a more physiologically accurate measurement that better reflects what actually resists digestion in the human small intestine (the physiological definition of dietary fiber), and has become the basis for dietary fiber values ​​on millions of nutrition labels worldwide.

This method uses a combination of enzymatic digestion and gravimetric determination:

• Sample preparation: Double test samples are gelatinized with heat-resistant α-amylase.
• Enzymatic digestion: Protease (to remove protein) and amyloglucose (to hydrolyze digestible starch into glucose) enzymes simulate human digestive conditions (pH, temperature, and duration similar to those in the mouth, stomach, and small intestine).
• Dietary fiber precipitation: Ethanol (4 volumes) is added to precipitate the soluble dietary fiber polymers and remove the digested substances.
• Filtering and washing: The residue (containing total dietary fiber) is filtered, washed with ethanol and acetone, dried, and weighed.
• Protein and ash correction: One pair of samples is analyzed for residual protein, the other for ash; these are subtracted to obtain the corrected total dietary fiber.
• Optional separation of soluble and insoluble fibers: Before ethanol precipitation, the digestive product can be filtered. The residue remaining after filtration = insoluble dietary fiber. The filtrate is then precipitated with ethanol to recover soluble dietary fiber. Total dietary fiber = Insoluble dietary fiber + soluble dietary fiber.

The basic characteristics of this method are as follows:

• It measures both soluble and insoluble dietary fiber.
• It contains resistant starch that is resistant to enzymatic digestion.
• DP contains most of the indigestible oligosaccharides, the lowest being the top 10 (because they precipitate in 78% ethanol).
• DP excludes indigestible oligosaccharides 3-9, which require separate methods.

The advantages of this method are as follows:

• Relatively simple equipment (no HPLC required).
• Robust and reproducible results across laboratories.
• Comprehensive validation data and inter-laboratory studies.
• It measures physiologically relevant fibers.

Current practical use:

• It is still used as a reference method in many countries that have not adopted the new Codex/FDA definition.
• It is frequently used in research when comparing historical data.
• New methods, where differences are minimal, are preferred by many laboratories for grain products, vegetables, and traditional high-fiber foods.

For many years, our organization, with its competent and experienced staff, has been understanding the needs of its clients, providing the advanced testing services they require, and assisting in the establishment, implementation, and improvement of management systems. Within this framework, we also provide testing services to businesses in accordance with the "AOAC 991.43 Total, soluble and insoluble dietary fiber in foods - Enzymatic-gravimetric method MES-TRIS Bufler A" standard.