Table 3 Methods of modified starch production




Physically Pregelatinization Starch paste is precooked modified and dried by extrusion or drum drying Dextrinization Starch polymers are hydrolyzed to smaller molecules by irradiation Chemically Derivatizationa Lateral groups are added modified to starch lateral chains

Cross-linkinga Multifunctional groups are used to link two different starch molecules together Dextrinization Starch polymers are hydrolyzed by oxidizing agents, acid hydrolysis, pyrodextrinization

Enzymatically Dextrinization Starch polymers are modified hydrolyzed to smaller molecules by incubation with amylases aDouble-derived starches are produced by combination of these two processes.

This complexity makes it difficult to quantify accurately. All in vitro methods therefore need to be corroborated against in vivo models; however, in vivo models are also very difficult to validate.

In general, in vitro methods try to imitate human small intestine digestion using different sample preparation (i.e., milling, chewing, etc.), sample pretreatment (i.e., simulation of oral or stomach digestion), sample treatment (i.e., different enzymes mixtures), sample post-treatment (i.e., different resistant starch solubilizing agents and enzyme mixtures), and incubation conditions (i.e., shaking/stirring, pH, temperature, time) (Table 4). The choice of each of these multiple factors represents a huge analytical problem because not only a compromise between physiological conditions and analytical handling has to be achieved, but also because the resistant starch content values must be in agreement with in vivo data.

On the other hand, in human in vivo methods, samples of digested food that reach the end of the small intestine are taken for analysis, either from ileostomy patients (i.e., where the large intestine has been removed) or from healthy volunteers using special cannulas in the ileum. Animals can also be employed for in vivo experiments, such as gnotobiotic (i.e., germ-free) and pseudognotobiotic (i.e., antibiotic treated) rats. In these cases, colonic bacterial fermentation is absent or suppressed by antibiotics and it is assumed that what reaches the end of the small intestine appears in feces. The main difficulty with these methods is that in vivo starch digestion may occur during the whole transit through the small intestine, which varies between individuals and the type of meal consumed. Moreover, these studies are difficult to perform in healthy volunteers and the physiological significance of using ileostomy patients is debatable, for example, it may not relate to infants and children who have decreased digestive capacity.

The initial in vitro assays were adapted from the enzymatic-gravimetric method used for dietary fiber assessment, but could only measure RS3. Soon new approaches to assess other types of resistant starch were developed. The Berry method, for instance, measures both RS3 and RS2 using an exhaustive incubation (16 h) of milled sample with a-amylase and pullulanase, following by centrifugation to separate the insoluble residue, which contains the resistant starch. This residue is treated with KOH to disperse retrograded and native starches, which are then hydrolyzed to glucose with amyloglucosidase. Finally, released glucose is quantified by a colori-metric assay. The Berry method has been subsequently modified by Faisant et al. and Goni et al.: the pullulanase was eliminated from the enzyme mixture and a pretreatment with pepsin added to decrease starch-protein interactions (Table 4).

Other methods have been developed to assess all types of resistant starch. Indeed, the Englyst method was developed to assess all nutritionally important starch fractions, such as rapidly digestible and slowly digestible starches, along with the three types of resistant starches described above. In this method, resistant starch fractions are estimated altogether by difference between total and digestible starches. Sample preparation is kept to a minimum in an attempt to mimic the way food is consumed. After pretreatment with pepsin, the sample is incubated with a mixture of amylogluco-sidase, invertase, and pancreatic enzymes for 2 h. Glucose released is then used to estimate the digestible starch. Next, total starch is measured as glucose released after solubilization of the nondigestible fractions with KOH, followed by amyloglucosidase hydrolysis. The Englyst method also allows evaluation of RS1, RS2, and RS3. The main problem with this method is its low reproducibility, especially between laboratories, because of the technical difficulties involved. Two other methods include chewing by volunteer subjects as sample preparation. In the Muir method, for instance, the chewed sample is sequentially treated with pepsin and an amyloglucosidase-pancreatic amy-lase mixture to obtain the nondigestible fraction, which is then boiled with Termamyl (a thermostable a-amy-lase) and solubilized with dimethyl sulfoxide followed by another amyloglucosidase-pancreatic amylase mixture step to yield finally glucose. The Akerberg method is similar to the Muir method, but it includes other

Table 4 Comparison between different methods to measure resistant starch in vitro



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