ANALYTICAL METHODS FOR RS

During recent years, a lot of effort has been put into the evaluation, then into the improvement of the existing methods of RS. Roughly, two analytical methods are now proposed in the literature: one was described by Englyst et al. (1992)(2), the other was obtained during the European Research Program EURESTA (6).

They provide very similar data for most starch samples however they both proba-bly underestimate RS as defined above. Indeed, RS collected in vivo in humans (ileostomates or healthy subjects using the intubation technique) is composed of oligosaccharides (including glucose), α-glucans of high molecular weight (mainly starch granules) and a crystalline fraction whose size depends on the origin and the treatment of the starch. None of the analytical methods of RS takes

Analytical Methods 173

into account the potentially digestible starch (oligosaccharides and part of the high molecular weight fraction) (7,8).

General Principle

In order to quantify RS, the first step is to remove the digestible starch from the sample. This is performed using a pancreatic (α-amylase. In some of the methods, an amyloglucosidase is added in order to avoid a possible inhibition of the α-amylase by the products of the digestion (mainly maltose, maltotriose). The amy-lolysis is also sometimes preceded by a proteolysis which is supposed to reflect the action of the pepsin inside the stomach and of the trypsin which is secreted in the pancreatic juice together with theα-amylase.

After the hydrolysis of the digestible starch, RS is quantified directly in the residue (isolated most of the time by 80% ethanolic precipitation) (6,9) or by difference between total starch and digestible starch which are quantified sepa-rately (2).

Main Analytical Methods

The main methods proposed in the literature will be briefly described and criti-cized.

The Method of Bjoˆrck et al., 1986 (10) Principle

It quantifies the starch residue present in the dietary fiber residue obtained by the methods of Asp et al. (1983) (11) or Prosky et al. (1988) (12).

Procedure (Figure 1) Main Characteristics

• Some of the samples such as bread can be analyzed as eaten without further drying. All the samples have to be milled (⬍0.5 mm).

• The samples are then submitted to a triple hydrolysis by Termamyl (100°C, 15 min), pepsin and pancreatin. The main consequence of this procedure is the gelatinization of all raw starch present in the sample which cannot be quantified as resistant starch.

Advantages and Drawbacks

• Can be performed with total dietary fiber analysis

• Analyze mostly retrograded starch

• Validated with a rat model (antibiotic treated rat). Figure 2 shows the

174 Champ et al.

FIGURE1 Determination of resistant starch according to Bjoˆrck et al., 1986 (10) (From Ref. 6).

good correlation between this in vitro method and the determination of the digestibility in antibiotic (Nebacitin) treated rats (13).

The Method of Englyst et al., 1992 (2) Principle

The various types of starch are determined by controlled enzymic hydrolysis and measurement of the released glucose using glucose oxidase. Resistant starch is defined as the starch not hydrolyzed after 120 min incubation with pan-creatic amylase and amyloglucosidase at 37°C. It is calculated by deducting from total starch content, the slowly digestible starch and the rapidly digestible starch contents of the sample hydrolyzed respectively after 120 and 20 min incubation.

Analytical Methods 175

FIGURE2 Correlation between in vitro method (10) and the determination of the digestibility in antibiotic (Nebacitin) treated rats (From Ref. 13).

Procedure (Figure 3) Main Characteristics

• Fresh foods can be analyzed as eaten. They are passed through a mincer.

The hydrolysis is performed in presence of glass ball and guar gum (to prevent mechanical alteration of very sensitive starch such as raw potato starch).

• The samples are hydrolyzed with pancreatin, amyloglucosidase and in-vertase. The pancreatin contains several enzymic activities including α-amylase and proteolytic enzymes which allows the digestion of the digestible material (starch and proteins). In contrast, the lipolytic activ-ity of the pancreatin might be ineffective due to the absence of bile in the medium, into glucose and fructose. The invertase is added in order to hydrolyze all the sucrose present in the sample. The sucrose must then be quantified separately. This step has been introduced in the method because of the presence of an invertase contaminating activity in the amyloglucosidase. This activity could lead to an overestimation of the starch content of the sample.

• The digestible starches (starches hydrolyzed after 20 and 120 min hy-drolysis) are quantified on an ethanolic extract (64.4% ethanol). The extract is hydrolyzed into glucose by amyloglucosidase. Then the glu-cose is quantified using GOD-PAP kit.

• RS content is determined by difference between total starch and digest-ible starch contents.

176 Champ et al.

FIGURE 3 Determination of resistant starch according to Englyst et al., 1992 (2).

Advantages and Drawbacks

• Optimization of the conditions of hydrolysis

• Adapted to a large variety of substrates

• Long and poor reproducibility without long training

• Need for very specific equipment (mincer, shaking water-baths)

• Enzymes not all commercially available

Analytical Methods 177

The Method of Muir & O’Dea, 1992 & 1993 (14, 15) Principle

The chewed food sample is incubated with pepsin, then hydrolyzed byα-amylase and amyloglucosidase. The insoluble residue represents RS.

Procedure (Figure 4) Main Characteristics

• The food is first chewed by a volunteer in standardized conditions. The sample weighed for the analysis contains about 100 mg carbohydrate.

• Pepsin,α-amylase and amyloglucosidase are used for the hydrolysis of the digestible fraction of the sample.

• RS is recovered by centrifugation followed by aqueous washing of the pellet.

• Starch in the residue is hydrolyzed into glucose by Termamyl then pan-creatin and amyloglucosidase. Dimethylsulfoxide is used prior to the second hydrolysis step to allow a complete dispersion of the RS. Glu-cose is then quantified using a GOD-PAP kit.

Advantages and Drawbacks

This method has been validated with in vivo studies on ileostomates (15,16).

From the 7 comparisons available, it seems satisfactory. No comparison with other in vitro have been published. Being used, until now, only by the authors, difficulties and reproducibility cannot be evaluated.

The Method of Champ et al., 1992 (6) Principle

In vitro RS was defined as the starch not hydrolyzed by incubation with α-amylase. Hydrolysis products are extracted in 80% ethanol and discarded. RS is then solubilized with 2N KOH and hydrolyzed with amyloglucosidase. In the new procedure, amyloglucosidase is added to the pancreaticα-amylase to avoid inhibition of the amylase by the products of the digestion.

Procedure (Figure 5) Main Characteristics

• In the first two methods, the sample has to be dried (freeze-dried when possible) and ground into fine particles. In the new procedure, fresh

178 Champ et al.

FIGURE 4 Determination of resistant starch according to Muir & O’Dea (14, 15).

samples can be analyzed after a grinding in a mincer which is supposed to simulate chewing of the food.

• 100 mg of test material are weighed for the analysis, in Faisant et al.

(1995)(9). In the new procedure, the sample must be weighed to contain about 50 mg starch.

Analytical Methods 179

FIGURE 5 Determination of resistant starch according to Champ (1992) (Method A) (From Ref. 6).

• Only pancreaticα-amylase is used for the hydrolysis of the digestible starch in the first two methods whereas amyloglucosidase is introduced in the new method. Sodium azide is used in the last two methods to prevent bacterial proliferation (and degradation) during the overnight hydrolysis.

• RS is recovered by ethanolic precipitation. In Champ method (1992)(6) the precipitate was washed then dried with acetone. The drying has been suppressed in the last two methods.

• RS is then dispersed using potassium hydroxide before the amyloglu-cosidase hydrolysis into glucose. A first step of gelatinization has been added in the last two methods to facilitate starch dispersion. Glucose is analyzed with GOD-PAP kit.

180 Champ et al.

Advantages and Drawbacks

• Simple and relatively rapid

• Ten samples can easily be analyzed (in duplicates) in a normal day of work

No particular training is needed. The first methods were optimized using values provided by Englyst et al. (2). The new procedure has recently been validated in collaboration with Dr. Langkilde and Prof. H. Andersson using in vivo values obtained from ileostomates. More comparisons will be performed between in vitro and in vivo values.