2017 Volume 23 Issue 2 Pages 339-342
We attempted to reduce the digestibility of potato starch by adding fatty acids (palmitic or linoleic acid) and treating with heat in order to develop a starch product with high consumer acceptability. The starch digestion rate was measured by enzymatically hydrolyzing starch samples using porcine pancreas α-amylase (EC 3.2.1.1) and amyloglucosidase (EC 3.2.1.3), and measuring the amount of glucose released after 90 min. In addition, we evaluated the starch digestion rate by calculating the in vitro glycemic index (EGI) using the method of Goni et al. (1997). The greatest EGI reduction (approximately 60% reduction compared to non-treated samples) was observed in potato starch samples adjusted to 20% moisture content and to which at least 3% fatty acid had been added. In this study, we were able to create a resistant potato starch by using simple food processing techniques of fatty acid addition and heat treatment.
Given their effectiveness in suppressing increases in postprandial glucose and blood insulin secretion, resistant starches (RS) have received attention for their potential to prevent diabetes (Granfeldt et al., 1995). The term RS was first used to refer to starches with the aforementioned physiologic function by Englyst et al. (1982). Brown et al. (1995) classified RSs into the following four categories: RS1 are physically enclosed within cell walls and are inaccessible to digestive enzymes; RS2 are raw starches that exhibit B-type X-ray diffraction; RS3 are retrograded starches that gelatinize and then recrystallize into a stable structure; and RS4 are processed starches, such as cross-linked starches (phosphate distarch phosphate) and highly etherified (hydroxypropyl starch) starch, whose resistance to digestion has been enhanced and remains enhanced even after food processing. However, as modified RS4 are considered to be food additives, information to that effect must be displayed on ingredient labels. Furthermore, special facilities are needed to manufacture modified starches, thereby driving up processing costs. Therefore, we attempted to confer digestion resistance to starch using simple food processing techniques, specifically by adding fatty acids and treating with heat, with the goal of developing a starch product with high consumer acceptability. The fatty acids used in this study were palmitic and linoleic acids, which are commonly found in vegetable fats and oils (Gomyo and Hasegawa, 1993).
Materials Potato starch (Calbee Inc., Tokyo, Japan) was used as the starch sample. The composition of the potato starch (per 100 g, approximately) was as follows: saccharides, 81.6 g; protein, 0.1 g; fats, 0.1 g; water, 18.0 g; and minerals, 0.2 g. Palmitic and linoleic acids (Wako Pure Chemical Industries Inc., Osaka, Japan) were used as the fatty acids. Porcine pancreas α-amylase (EC 3.2.1.1) and amyloglucosidase (AMG) (EC 3.2.1.3) from a Resistant Starch Assay Kit (K-RSTAR; Megazyme Inc., Bray, Ireland) were used as hydrolytic enzymes.
Sample preparation Moisture content of the potato starch was adjusted to 11 or 20%, and fatty acids (palmitic, linoleic acids) were added to the starch to achieve 0, 3, 5 or 7% content (w/w). Next, the starch samples were sealed in airtight stainless-steel containers and heated in an oil bath until the internal temperature of the samples reached 140°C. After the target temperature was achieved, samples were rapidly cooled in ice water and used for measurement. Glucose content was expressed per gram of sample (dry basis).
Measurement of starch digestion rate by enzymatic hydrolysis The starch digestion rate was evaluated by monitoring the amount of glucose released after 90 min. After adding 15 mL of 0.1 M maleic acid buffer solution (pH 6.0) containing 0.003 M CaCl2 to 75 mg of sample, the resulting slurry was heated for 60 min at 100°C. After cooling the samples to 37°C, 0.069 U of porcine pancreas α-amylase (Ceralpha Method, 3 Ceralpha units/mg) was added, and the samples were incubated for 90 min at 37°C with shaking (100 rpm). Aliquots (0.4 mL) of the samples were collected into microtubes. To deactivate α-amylase, samples were immediately heated for 5 min at 100°C. Next, samples were centrifuged (11752 × g, 5 min), and 0.1 mL of the supernatant was collected. After adding 0.37 mL of 0.4 M sodium acetate buffer solution (pH 4.5) and 0.1 U of AMG (3300 U/mL on soluble starch) to the supernatant, the resulting solution was shaken (85 rpm) for 45 min at 60°C to cleave glycoside linkages. Glucose was measured using the glucose oxidase/peroxidase method. Measurements were performed three times for each sample. For statistical analyses, t-tests (for two samples assuming equal variances) were performed using Microsoft Excel 2010.
Calculation of estimated glycemic index (EGI) The EGI was calculated following the digestion model method based on enzymatic digestion by Goni et al. (1997). Relative digestion rates (Hi) were calculated comparing sample digestion rates to the white bread (reference foodstuff) digestion rate using Eq. 1 below. The H90 for white bread represents the mean for three commercially-available products.
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Finally, EGI was calculated using Eq. 2 proposed by Goni et al.
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Effects of fatty acid type and addition rate on digestibility We evaluated the digestibility of the samples by measuring the glucose content of samples 90 min after enzyme addition. The effects of potato starch moisture content, fatty acid type, and fatty acid addition rate on digestibility are shown in Figs. 1 and 2.
Effects of potato starch moisture ocntent and palmitic acid addition on digestibility. Samples were enzymatically digested in vitro for 90 min.
Glucose content is expressed per gram of sample(dry basis).
*Significantly different vs. untreated control sample at P<0.05.
Control 
Palmitic acid 3% 
Palmitic acid 5% 
Palmitic acid 7%
Effects of potato starch moisture content and linoleic acid addition on digestibility. Samples were enzymatically digested in vitro for 90 min.
Glucose content is expressed per gram of sample (dry basis).
*Significantly different vs. untreated control sample at P<0.05.
Control Linoleic acid 3% Linoleic acid 5% Linoleic acid 7%
With regard to the effects of fatty acid type, for samples adjusted to 11% moisture content, greater reduction of digestibility was observed in samples treated with linoleic acid than those treated with palmitic acid. However, for samples adjusted to 20% moisture content, little difference was observed in the digestibility of samples, regardless of the type of fatty acid added. With regard to fatty acid addition rate, for samples adjusted to 11% moisture content, digestibility tended to decrease with increasing fatty acid addition rate for both fatty acid types. In contrast, for samples adjusted to 20% moisture content, although slight differences in digestibility were observed in samples treated with palmitic acid, no clear relationship was observed between fatty acid addition rate and digestibility. Similarly, no clear relationship was observed between the linoleic acid addition rate and digestibility. With regard to the effects of moisture content, both in the case of samples treated with palmitic acid and those treated with linoleic acid, substantially greater reductions in digestibility were observed in samples adjusted to 20% moisture content when compared to those adjusted to 11%. No comparable reduction in digestibility was observed in non-treated samples.
The greatest reduction in digestibility was observed in samples adjusted to 20% moisture content and to which at least 3% fatty acid had been added.
Calculation of EGI In order to evaluate starch digestion rates, we calculated EGI based on the results shown in Figs. 1 and 2 according to the method by Goni et al. (1997). The results of these calculations are shown in panels A and B of Fig. 3. In potato starch adjusted to 11% moisture content (panel A), EGI tended to decrease, albeit slightly, with increasing fatty acid addition rate. In addition, significant differences were observed between fatty-acid treated samples and untreated controls. In particular, the EGI of linoleic acid-treated samples decreased by 30 to 40% relative to untreated controls.
Effects of potato starch moisture content and fatty acid type and addition rate on EGI. EGI for moisture content adjusted to 11%(panel A) or 20%(panel B). *Significantry different vs. untreated control sample at P<0.05.
Linoleic acid Palmitic acid Control
In contrast, in potato starch adjusted to 20% moisture content (panel B), increasing the fatty acid addition rate did not affect EGI. However, EGI was reduced dramatically (by at least 60% relative to untreated controls) in samples to which at least 3% fatty acid had been added.
Comparing panels A and B, the fact that the EGI of the untreated control essentially does not change suggests that the addition of both water and fatty acid substantially changes EGI.
The addition of fatty acid was confirmed to reduce EGI relative to the untreated controls (Fig. 3). This reduction is believed to primarily result from the formation of complexes between the starch and the fatty acid. With regard to the mechanisms of complex formation, straight-chain saturated fatty acids such as palmitic acid penetrate the amylose helix structure to form fatty acid-amylose complexes. As a result, hydrophobic regions are created along the starch chain. Schoch (1942; 1944) and Mikus et al. (1946) reported that fatty acids in starch granules are contained within amylose helices, supporting the results of this study. In contrast, unsaturated fatty acids such as linoleic acid contain double bonds; the resulting kinked molecular structure is believed to sterically hinder fatty acids from penetrating the amylose helix structure. However, similar reductions in EGI were observed in samples treated with linoleic acid. Akuzawa et al. (1995; 1997) reported that although linoleic acid does not form complexes within amylose, it coexists with amylose chains and is subsumed within starch. Another possible mechanism has to do with the presence of relatively larger numbers of phosphate groups on the surface of potato starch compared to other starches, which may enable unsaturated fatty acids such as linoleic acid to form phosphate ester bonds. As a result, hydrophobic regions may be formed along the starch chain. In other words, we speculate that the formation of complexes within and on the outer surface of the amylose helices of potato starch keep α-amylase from approaching the starch chain.
According to Takaoka and Nikuni (1952), fatty acids with relatively short carbon chains, such as caprylic and capric acids, readily form complexes with amylose. However, Maezawa et al. (1968) reported that such fatty acids, even when incorporated into amylose, are easily extracted with ethyl ether. It is for this reason that we used palmitic and linoleic acids, which are found at high levels in vegetable fats and oils, in this study. As a result, we were able to create a resistant starch using simple food processing techniques; specifically, by adding fatty acid to potato starch and treating with heat. Reduction of starch digestibility through the addition of vegetable fats and oils is a practical approach from a food-processing standpoint. Accordingly, the next step will be to identify fats and oils that effectively confer resistance to starch. In the context of food safety, the oxidative stability of linoleic acid upon heating should be evaluated in future work.
