Analyzing Resistant Starch

Resistant starch may be a really hot topic only in the eyes of those who are selling it, and a hot topic only among those doing research on its functionality and potential nutrition benefits, purports Jonathan DeVries, Technical Manager at Minneapolis, Minn.–based Medallion Laboratories Div. of General Mills. But despite one’s enthusiasm or lack of it for this increasingly significant dietary component, the ongoing global debate over what specifically constitutes resistant starch is undeniable, he says.

For many years, DeVries has been involved with international efforts regarding the definition and appropriate laboratory analysis procedures for dietary fiber (see, for example, Devries and Rader, 2005).

"As scientific knowledge about the formation, chemistry, and physiology of resistant starch has increased throughout the world in recent years, not to mention regulatory changes in food ingredient labeling, the definition of this carbohydrate subset has continued to evolve," DeVries points out.

Resistant starch has been defined as that fraction of dietary starch that escapes digestion in the small intestine of healthy individuals. Because it is not digested, it functions as dietary fiber and provides several well-documented benefits relative to intestinal and colonic health, glycemic management, weight management, and energy.

It has been measured using standard dietary fiber methodology. It has also been measured chemically as the difference between total starch (TS) measured as glucose released from a homogenized and chemically treated sample and the sum of the glucose released from rapidly digestible starch (RDS) and that released from slowly digestible starch (SDS) during enzyme digestion of non-homogenized food samples:

RS = TS – (RDS + SDS)

"Confusion stems in large part from the fact that resistant starches are divided somewhat arbitrarily into four categories—RS1, RS2, RS3, and RS4," DeVries says.

RS1 is starch physically trapped within the matrix of food, such as starch granules locked in the plant cell by the cell wall. Typically found in seeds, legumes, and unprocessed grains, RS1 is enzyme resistant simply because amylolytic enzymes have no physical access to it. This type of resistant starch is affected largely by chewing and by food processing steps such as grinding and milling.

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Resistant starch that occurs in its natural granular form is classified as RS2. In raw starch granules, starch is tightly packed in a radial pattern and is relatively dehydrated. This compact structure limits the accessibility of digestive enzymes and accounts for the resistant nature of RS2, such as ungelatinized starch. Examples include uncooked potato, green-banana flour, and high-amylose grains. The enzyme resistance of RS2 can be overcome by gelatinization.

Gelatinized and retrograded starch constitute the RS3 category. Cooked and cooled potato starch, enzymatically debranched starches, and cooled starches in cooked products (e.g., some sauces and dips) hold the RS3 distinction.

Modified starches obtained by chemical treatments like distarch phosphate ester are classified as RS4.

Complications with Resistant Starch Analysis
Starches that are highly resistant to digestion are generally determined as dietary fiber in the standard dietary fiber analysis. AOAC Official Method 2002.02, "Resistant Starch in Starch and Plant Materials," is clearly available for analysis of resistant starch. "However," DeVries says, "this method is applicable to plant and starch materials, primarily types RS2 and RS3, but is not applicable to food products. Therefore, we currently are limited to the use of standard dietary fiber methodology to determine the total dietary fiber content of a food product. The resistant starch will be included in that total dietary fiber quantity."

Moreover, it’s technically possible that the resistant starch content of a food will increase or decrease as a result of processing conditions such as pH, heating temperature and time, number of heating and cooling cycles, freezing, and drying.

"In some cases, the resistance to digestion of resistant starch is lost during processing," DeVries continues. "For example, raw potatoes are very high in resistant starch. When the starch is gelatinized with heat, it becomes very digestible. However, on cooling, a portion of the digestible starch retrogrades and becomes resistant to digestion. Thus, when a resistant starch ingredient has been formulated and processed into a product, the quantity of dietary fiber found analytically may not match the quantity of dietary fiber calculated on the basis of ingredient analyses and formulation. As a result, one must analyze the final product for an accurate determination of total dietary fiber."

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Steps in Analysis
According to work conducted by McCleary and Rossiter (2004), the main step of any method to measure the content of resistant starch in foods must be to first remove all of the digestible starch from the product using thermostable α-amylases. At present, the method developed by McCleary and Monaghan (2002) is considered the most reproducible and repeatable measurement of resistant starch and plant materials, but it has not been shown to analyze all resistant starch as defined by Champ et al. (2003). Two general methods specifically proposed to determine resistant starch—Berry (1986) and Englyst et al. (1992)—remove digestible starch using different amylases, and the residual fraction is quantified after solubilization in 2M KOH.

The Siljestrom and Asp (1985) procedure includes preparation and quantification of dietary fiber residue before resistant starch determination. This is usually done by drying the samples at 100°C. Since heating influences the resistant starch content in foods, results may be modified by this step.

A modified method developed by Saura-Calixto et al. (1993) for measuring resistant starch in dietary fiber residues from various sources involves mixing fiber residues with KOH, acetate buffer, and HCl. After incubation with amyloglucosidase, samples are centrifuged and diluted with distilled water. Resistant starch is calculated as milligrams of glucose × 0.9. Advantages of this method are the use of small amounts of sample, less reagents, and elimination of drying.

All determinations of resistant starch as dietary fiber are currently carried out using traditional laboratory procedures. "There are no special instruments or equipment available or needed for the enzymatic, gravimetric methods," DeVries says. "Regardless of where in the world the analyses for total dietary fiber—which will include the resistant starch—are done, the tests are conducted manually."

When a given amount of resistant starch is added to a recipe, the amount that remains resistant may increase or decrease by the time the food is processed into a finished product, he points out. So it’s not possible to calculate fiber based on percentage of resistant starch added into a product.

"My recommendation is to use AOAC methods 985.29 and 991.43 for dietary fiber analysis and the resistant starch content," DeVries says.

What does the future hold for resistant starch? "That largely depends on how successfully it is marketed and competes with other ingredients," he emphasizes. "However, I believe resistant starch will continue to be added to foods to increase fiber content because it’s difficult for most people to consume the recommended 25–35 g of dietary fiber per day by consuming a normal diet. And the debate will continue on what exactly constitutes resistant starch."

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Focus on Fiber
While controversy clouds methods for classifying and determining resistant starch content in foods, the fiber common denominator smoothes troubled waters worldwide, according to Rhonda Witwer, Business Manager, Nutrition at National Starch Food Innovation, Bridgewater, N.J. The company developed and markets Hi-maize 5-in-1 Fiber, a high-amylose corn that is billed as the only commercially available natural RS2 resistant starch.

Whereas natural sources of resistant starch found in foods such as potatoes are subject to change, making them unreliable as a fiber source, she says, Hi-maize is advantageous in foods on account of its stability.

Hi-maize tests as an insoluble fiber. According to Witwer, its physical properties, particularly its low water-holding capacity, enable it to provide good food processing characteristics and desirable textural attributes, compared to foods of similar fiber content.

"Those of us in communications can come to an understanding based on fiber content," Witwer relates. "If a component tests like fiber and is labeled as fiber, we can have meaningful communication in the fiber realm."

It’s difficult to differentiate resistant starches in various plants due to individual components and effects that can occur within a specific category, Witwer points out. "And if there’s a question about whether or not an item even contains resistant starch, we come back to the physiological evidence, because we have a lot of it," she says.

Aunt Millie’s bakeries (formerly Perfection bakeries, Inc.), Fort Wayne, Ind., has been incorporating Hi-maize 5 in 1 Fiber in various formulations with other fibers since 2003, according to Rod Radalia, Director of Technical Services for the firm.

"We use Hi-maize to add fiber to our products and reduce calories in them," Radalia says. "Tests for the percentage of resistant starch in the finished product are done by mathematical calculations. After adjusting for a 10% moisture loss during baking, the final product will have an amount of resistant starch proportional to the batch weight."

"I’m excited about the health benefits and the research behind Hi-maize resistant starch," Radalia adds. "Consumer demand will continue to drive the popularity of resistant starch throughout the industry. Physically active young adults increasingly want the benefits resistant starch offers, as do empty-nesters who want to maintain their health as long as possible."

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