Quantification of Resistant Starch in Several Starch Sources Treated Thermally

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Quantification of Resistant Starch in Several Starch Sources Treated ThermallyL. A. Brumovsky a; J. O. Brumovsky a; M. R. Fretes a; J. M. Peralta ab

a Facultad de Ciencias Exactas Químicas y Naturales, Universidad Nacional de Misiones, Posadas, Misiones,Argentina b Instituto de Desarrollo Tecnológico para la Industria Química (INTEC). CONICET-UNL, Santa Fe,Argentina

Online Publication Date: 01 July 2009

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International Journal of Food Properties, 12: 451–460, 2009Copyright © Taylor & Francis Group, LLCISSN: 1094-2912 print / 1532-2386 onlineDOI: 10.1080/10942910701867673

451

QUANTIFICATION OF RESISTANT STARCH IN SEVERAL STARCH SOURCES TREATED THERMALLY

L.A. Brumovsky1, J.O. Brumovsky1, M.R. Fretes1, and J.M. Peralta1,2

1Facultad de Ciencias Exactas Químicas y Naturales, Universidad Nacional deMisiones, Posadas, Misiones, Argentina2Instituto de Desarrollo Tecnológico para la Industria Química (INTEC). CONICET-UNL, Santa Fe, Argentina

The purpose of the research was to determine the effect of heat-moisture treatment at differentphases and temperatures on resistance starch (RS) level on native starches samples of severalbotanical sources and to evaluate the thermal stability of different granules. Samples ofpotato, cassava, wheat, and corn starches were moisturized up to 30% wet basis and thentreated in a convection oven at 80, 100, and 120°C during 40 and 60 minutes. RS was deter-mined gravimetrically by a modification of Method 991.43 of the AOAC for the determination oftotal dietary fibre. All samples were submitted to different thermal analysis in a range from40 to 180°C at 10°C/min. Overall, in the treated samples an increase of RS was observed,being the corn starch sample treated at 120°C and 60 minutes – the one that presented thehighest content of RS (4.2%). Other treated samples showed a decrease in the gelatinizationenthalpies with the presence of granular fusion, indicating internal re-arrangement, anincrease in the gelatinization temperature and the thermo stability below 95°C.

Keywords: Resistant starch, Heat-moisture treatment, Hydrothermal treatments.

INTRODUCTION

Resistant starch (RS) has been defined as “the sum of starch and products of starchdegradation not absorbed in the small intestine of healthy individuals”.[1] Even though RSescapes digestion in the small intestine, it may be fermented in the large intestine by thecolonic microflora. [2,3,4,5]

In the last decade, there has been an increased interest in the nutritional implicationsof RS, not only for its low caloric content, but also because it may have a physiologicaleffect similar to that dietary fibre.[6,7,8] Moreover, the fact that processing treatments maymodify the RS content in foods has gained the attention of food technologists.[7,9,10]

RS has been classified as Type I, which results from the physical inaccessibility ofenzymes in intact tissues. Type II comes from the physical structure of native starch granules.Type III results from the retrogradation and gelatinization of starch in resistant structures

Received 1 April 2007; accepted 16 December 2007.Address correspondence to L.A. Brumovsky, Facultad de Ciencias Exactas Químicas y Naturales,

Universidad Nacional de Misiones, Félix de Azara 1552 – (3300) Posadas, Misiones, Argentina. E-mail: [email protected]

Downloaded By: [Brumovsky, J.O.] At: 13:54 4 May 2009

452 BRUMOVSKY ET AL.

to the digestive enzymes. Type IV is where the resistance results from the chemical modi-fication, which interferes with the enzymatic digestion.

Although several analytical in vitro methods have been developed to determineRS,[11,12,13,14,15] according to the physiologic definition, it cannot be established as invitro. Nowadays, there is no accepted analytical procedure to determine RS. This repre-sents an important limitation to evaluate RS production processes. The most commonlyused method for RS determination[16,17,18,19,20,21] is based on the AOAC method for themeasurement of total dietary fibre (TDF).[16] The TDF method detects the undigestedstarch remaining after 30 min of boiling with thermally stable a-amylase. This method,which was not initially intended for RS determination, detects only the fraction of RS,which survives a boiling treatment. The portion of RS isolated by the TDF method isimportant because it is not only appears as dietary fibre, but it also is relatively ther-mally stable because it remains after a thermal processing at 100°C. Although none ofthe analytical procedures are intended to evaluate the thermal stability of RS, thermallystable RS can be evaluated either by the Englyst method after a boiling step or by theTDF method.[22]

It is known that the quantity of RS can be increased by maintaining the structure ofthe granule. The addition of commercial RS to foods has allowed food technologists toproduce foods with dietary fibre without the fibre flavour, and to improve the functionalityin the alimentary processes. To produce commercial RS, the starches are exposed to acombination of biochemical and/or hydrothermal modifications. The biochemical modifi-cations require the use of enzymes or acids to remove the non-resistant parts of the starch.The hydrothermal modifications are treatments combining heat and moisture. Because theeffects of biochemical and hydrothermal modifications in the yield of RS are not wellunderstood, the production of RS is based on empirical methods, and consequently thepotential is not known to produce RS. Jacobs and Delcour[23] define a hydrothermal modi-fication as any treatment of heat and moisture carried out at temperatures above the glasstransition temperature but below the gelatinization temperature of starch, without destroyingthe granule structure.

The water content to which the starch is treated by heating is important to determineits physiochemical characteristics. Some authors[23,24,25] use the term “annealing” referringto a treatment of heat with high moisture (> 40% wet basis), and “heat-moisture treatment”referring to a heat treatment of at low moisture (HMT) (< 35% wet basis). Moreover, itwas also demonstrated that the hydrothermal treatments on the granules of starch increaseor decrease the susceptibility to hydrolysis with a- amylase. These effects are related tothe botanical source[26,27,28] and to the conditions of treatment.[29,30]

In all thermal treatments, the thermal stability of the granules of starch depends onthe temperature and the content of water where these treatments are applied. Therefore,when the conservation of the granular structure is sought, there are conditions of tempera-ture and critical content of water, over which the thermal treatments destroy the structureof the granule, and consequently they should not be exceeded. On the other hand, the rela-tionship between the temperature and water content to which the thermal treatmentsdestroy the granular structure is complex, depending on the source of starch. However, forcertain conditions the temperature and water content can be determined experimentally,for example, by means of differential scanning calorimetry. It is known that more energeticmodifications of the structure of the granule are achieved when the hydrothermal modifi-cations are carried out at temperatures and water contents next to the critical conditions,over which the structure of the granule is lost.


RESISTANT STARCH TREATED THERMALLY 453

In the annealing, the starch granules are treated at temperatures below of their gela-tinization temperature and with an excess of water. Although there are many combinationsof temperature and water contents through which the thermal treatment would correspondto an annealing treatment, the most energetic and quick modifications happen near gelati-nization temperatures. In a similar way to the annealing treatment, the heat-moisture treat-ment involves a thermal treatment of the starch granules below the fusion temperature,where the structure of the granule is lost in reduced water contents and at low content ofwater. The purpose of this research was to determine the effect of heat-moisture treatmentat different phases and temperatures on RS level on native starches samples of severalbotanical sources and to evaluate the thermal stability of different granules.

MATERIALS AND METHODS

Starches Samples

Starch samples used in this research were potato starch (Number 4251 catalogue,Sigma, St. Louis, MO), wheat starch (Number 5127 catalogue, Sigma, St. Louis, MO),corn starch (Number 5296 catalogue, Sigma, St. Louis, MO), and commercial cassavastarch obtained from VALOIS SACIFIA, Gdor. Roca, Misiones, Argentina.

Physiochemical Characterization

The gelatinization and fusion temperatures were determined by differential scanningcalorimetry using starch suspensions of 30% (w/w) and 70% (w/w), respectively. Thermalanalysis was performed using a differential scanning calorimeter (DSC 7, PerkinElmerCorp., Norwalk, CT) equipped with a thermal analysis data station (PerkinElmer). Indium(m.p. 156.4 °C, ΔHm 28.45 J/g) was used as a calibration standard. The reference cell con-tained a sealed, empty, stainless steel pan. Starch samples (∼15.0 mg, dwb) were weighedinto preweighed stainless steel pans (Perkin-Elmer). Deionized water was added to obtain∼ 30% (w/w) or ∼ 70% (w/w) starch suspensions. The samples were stirred with a needle.Pans were sealed, the total weights were determined, and the suspensions were storedovernight at room temperature. All samples were heated at 20°C up to 180°C, at a heatingrate of 10°C/min. Samples were then quench cooled from 180oC to 20oC at 10oC/min.Gelatinization enthalpy and peak temperatures were calculated (7 Series Software, Perkin-Elmer). Analyses were performed in triplicate and mean values and standard deviation arereported.

Hydrothermal Treatments

Considering initial fusion temperatures of the starches, the samples indicated inTable 1 were prepared adding deionized water from their initial moisture content up to30% w/w wet basis. In order to homogenize the distribution of water inside the granules,they were maintained at 25°C during 72 hours. Using a 22 ml stainless steel pressure vessel(model 4701 model, provided by Parr Instrument Co.), approximately 20 g of the hydratedsamples were thermostatized in a gravitational convection oven (± 1°C). Immediatelyafter the treatment, the vessel was submerged in a bath of water-ice during 5 min. Then thesamples were extracted and dried off in forced air oven at a temperature of 40°C until con-stant weighed. Then, they were sifted using a 120-mesh sieve (125 μm opening).



Boiling-Stable Resistant Starch Determination

The assay is based on the 991.43 method of the AOAC for the determination of totaldietary fibre.[16] The method was adapted for essentially ash and protein-free starch analysis.Starch samples (0.5 g) were suspended in 20 ml MES/TRIS buffer (pH = 8.2) and incu-bated with 25 μl of thermo stable a-amylase (Megazyme E-BLAAM. Wicklow, Ireland) at95°C−100°C during 35 minutes. The samples were then cooled at 60°C and adjusted topH 4.1–4.8 with 5% HCl or 5% NaOH. The samples were then incubated with 100 μl ofamyloglucosidase (Megazyme E-AMGDF. Wicklow, Ireland) at 60°C for 30 minutes.Mixtures were vacuum filtered with a #42 filter paper and washed with 10 ml of deionisedwater, 95% ethanol, and acetone. The boiling-stable portion of RS was determined as theresidue remaining after drying the samples in a convection oven at 103°C overnight.

Statistical Analysis

Statistical analysis was made using Minitab 13.20 (Minitab Inc., State College, Pa.,USA). To compare values of temperature and gelatinization energy, an ANOVA test wasused. To compare individual means an a posteriori test (LSD) was used (95% of signifi-cance level).

RESULTS AND DISCUSSION

Table 2 shows the boiling-stable RS content obtained following the pattern of theAOAC method for the determination of total dietary fibre[16] for the different samples ofnative and hydrothermal modified starches. A significant increase in the corn starch sample120ºC-60 min in relation to the native was found; but on the other hand, the other samplestreated did not present significant difference. Wheat starch samples treated during 60 minhad a statistically significant increase in relation to the native, without differences among

Table 1 Conditions of heat-moisture treatment (HMT) studied.

Samples Temperature (°C) Time (min)

Corn 100 40Corn 100 60Corn 120 40Corn 120 60Wheat 80 40Wheat 80 60Wheat 100 40Wheat 100 60Cassava 100 40Cassava 100 60Cassava 120 40Cassava 120 60Potato 100 40Potato 100 60Potato 120 40Potato 120 60



them. In addition, no significant differences were observed between the treatments of 40min and the native. The treatments at 100ºC caused no significant increase in the samplesof starch cassava, but on the other hand, a significant decrease was observed in the sam-ples treated at 120ºC in relation to the native. In potato starch significant differences werefound in three treatments (100ºC-60 min; 120ºC-40 min and 120ºC-60 min) in relation tothe native one and also among the samples at 120ºC-40 min and at 120ºC-60 min.

Relatively high variation coefficients (up to 27%) were obtained due to the low RScontent. Overall, the RS determinations showed an increase in the samples treated in rela-tion to their corresponding native starch, except for cassava starch which had a decrease(see Figure 1).

Ghua and Ali[31] suggested that the time and severity of thermal processing employedmay imparts specific properties to the respective starch-based products due to a partial orcomplete destruction of crystalline structure starch and considerable macro-moleculardegradation. On the other hand, RS overall resistance could be dependent of the structureof the starch granule.[32] Thus, the different RS contents found in the natives and treatedsamples could be due to the particular gelatinization process of each type of granule tothermal treatments.

The endotherms of the corn, wheat, cassava and potato samples (native andtreated) are shown in Figure 2. The corn endotherm presents its characteristic of main peakaround 65°C[33,34] and also a second component associated with melting of amylose-lipidcomplexes.[35] The treatments at 100°C tended to increase the peak of the main componentof endotherm with light loss of the gelatinization enthalpy (see Table 3). Treatments at120°C produced a partial fusion of the granule, being the most intense at 60 min, as can beobserved in the endotherms of Figure 2a. The wheat endotherm presents its characteristic

Table 2 Boiling-stable resistant starch contenta.

Starch Sample RS %b CV %

Corn Native 1.3 ± 0.1 7.7100ºC and 40 min 1.5 ± 0.3 20.0100ºC and 60 min 1.5 ± 0.2 13.3120ºC and 40 min 1.8 ± 0.1 5.6120°C and 60 min 4.2 ± 0.3 7.1

Wheat Native 1.0 ± 0.2 20.080°C and 40 min 1.1 ± 0.3 27.080°C and 60 min 1.9 ± 0.2 10.5100°C and 40 min 1.0 ± 0.2 20.0100°C and 60 min 1.6 ± 0.3 18.8

Cassava Native 1.1 ± 0.1 9.1100°C and 40 min 1.3 ± 0.2 15.4100°C and 60 min 1.3 ± 0.2 15.4120°C and 40 min 0.8 ± 0.2 25.0120°C and 60 min 0.8 ± 0.1 12.5

Potato Native 1.1 ± 0.2 18.2100°C and 40 min 1.1 ± 0.1 9.1100°C and 60 min 2.6 ± 0.5 19.2120°C and 40 min 2.4 ± 0.4 16.7120°C and 60 min 3.1 ± 0.6 19.4

a% w/w dry matter. bMean ± standard deviation of three to four replicates.



of main peak around 57°C[34] and a second component associated with melting of amylose-lipid complexes.[35,36] The treatments at 80°C tended to increase the peak of the main com-ponent of the endotherm with partial loss of the gelatinization enthalpy (see Table 3). Thethermal treatments at 100°C produced partial fusion of the granule (see Figure 2b), whichwas verified with the microscope of polarized light. The cassava endotherm presents itscharacteristic of an only peak around 63°C.[34,37] The treatments at 100°C tended toincrease the peak of the endotherm with light loss of the gelatinization enthalpy (see Table 3).The treatments at 120°C produced an important fusion of the granule, as can be observedin the endotherms of Figure 2c. The destruction of the granule due to the fusion was veri-fied with the microscope of polarized light. The endotherm of potato starch presents itscharacteristic of an only peak around 61°C.[33] The treatments at 100°C rose the peak ofthe endotherm to around 72°C (see Figure 2b). The thermal treatment at 100°C and 60 minshows fusion principle due to the appearance of two peaks in the endotherm. This suggeststhe formation of two types of starch crystallites.[33,38] Treatments at 120°C produced a par-tial fusion of the granule as can be observed in the endotherms of Figure 2d. The partialdestruction of the granule due to the fusion was verified with the microscope of polarizedlight.

The heat-moisture treatments studied caused an increase in gelatinization endot-herms width, with significant reduction of the gelatinization enthalpy (see Table 3), whatwas also observed by several authors[23,39,40]; and an increase in the gelatinization temper-ature, except for the corn starch sample treated at 120ºC and 60 minutes, (see Figure 2).Perera et al.[41] postulated that an increase in the peak temperature is a reflection of meltingof crystallite, which are formed because of amylase-amylose interaction and amylase-amylopectin interactions along the chains, which is stronger in heat moisture treatments.This suppresses the swelling of the granule leading to delayed gelatinization and a high onset,peak and conclusion temperature. The reduction in the gelatinization enthalpy following

Figure 1 RS average content in samples of native and hydrothermally modified starches.

1.1

2.62.4

3.1

0

1

2

3

4

5

Native 100–40 100–60 120–40 120–60

RS

% (

w/w

dry

mat

ter)

1.3 1.51.8

4.2

0

1

2

3

4

5

Native 100–40 100–60 120–40 120–60

RS

% (

w/w

dry

mat

ter)

1 1.1

1.9

1

1.6

0

1

2

3

4

5

Native 80–40 80–60 100–40 100–60

RS

% (

w/w

dry

mat

ter)

1.1 1.30.8

0

1

2

3

4

5

Native 100–40 100–40

–

120–40 120–60

RS

% (

w/w

dry

mat

ter)

WheatCorn

Cassava Potato

1.5

0.81.3 1.1



Table 3 Temperature of peak and gelatinization enthalpy.

Samples Peak temperaturea (°C) Gelatinization enthalpya (J/g)

Corn (native) 65.4 ± 0.56 21.1 ± 1.26Corn 100°C and40 min 76.0 ± 0.40 19.1 ± 0.51Corn 100°C and 60 min 77.5 ± 0.28 18.0 ± 0.15Corn 120°C and 40 min 77.5 ± 1.55 15.5 ± 7.03Corn 120°C and 60 min 54.5 ± 3.99 10.6 ± 1.53Wheat (native) 57.6 ± 0.14 24.8 ± 4.06Wheat 80°C and 40 min 69.7 ± 1.42 15.9 ± 0.58Wheat 80°C and 60 min 71.4 ± 0.48 15.1 ± 1.11Wheat 100°C and 40 min 72.3 ± 2.68 16.5 ± 3.57Wheat 100°C and 60 min 74.7 ± 0.94 16.1 ± 0.59Cassava (native) 63.4 ± 0.14 21.6 ± 1.54Cassava 100°C and 40 min 72.3 ± 0.02 17.9 ± 2.55Cassava 100°C and 60 min 74.3 ± 0.02 18.4 ± 0.75Cassava 120°C and 40 min 75.8 ± 0.68 7.6 ± 2.00Cassava 120°C and 60 min 74.6 ± 14.8 6.9 ± 0.59Potato (native) 60.7 ± 0.07 23.2 ± 0.13Potato 100°C and 40 min 71.6 ± 0.80 23.1 ± 1.04Potato 100°C and 60 min 72.6 ± 0.33 23.0 ± 2.53Potato 120°C and 40 min 68.5 ± 1.15 18.2 ± 0.47Potato 120°C and 60 min 69.0 ± 9.50 15.6 ± 4.45

aMean ± standard deviation of three replicates.

Figure 2 Endotherms of the starch samples.

20 40 60 80 100 120 140 160 180

Temperature (°C)20 40 60 80 100 120 140 160 180

Temperature (°C)

20 40 60 80 100 120 140 160 180

Temperature (°C)20 40 60 80 100 120 140 160 180

Temperature (°C)

En

do

ther

mic

hea

t fl

ow

(m

W)

En

do

ther

mic

hea

t fl

ow

(m

W)

En

do

ther

mic

hea

t fl

ow

(m

W)

En

do

ther

mic

hea

t fl

ow

(m

W)

120°C–60min 100°C–60min

100°C–40min

80°C–60min

80°C–40min

120°C–60min

120°C–40min

100°C–60min

100°C–40min

120°C–40min

100°C–60min

100°C–40min

Native Native

Native

120°C–60min

120°C–40min

100°C–60min

100°C–40min

Native

10 mW 10 mW

10 mW10 mW

Corn Wheat

Cassava Potato



hydrothermal treatment indicated partial gelatinization of some less heat stable mole-cules.[38] Hoover and Manuel[28] claimed that amylase content and starch chain lengthwere two significant factors determining the physical properties of the heat moisturetreated products. Not only the complex formation between amylose chains and otherstarch chains (mainly in amorphous regions), may cause physical changes in the treatedstarches. The structural difference among the amylose chains of the samples and the pres-ence of lipids in corn and wheat starches could result into the different influences by theheat moisture treatment upon the thermal characteristics of the starches.

CONCLUSIONS

The heat-moisture treatments studied in all the starches treated caused an increaseof the width of the gelatinization endotherms, with significant reduction of the gelatini-zation enthalpy, with the presence of granular fusion indicating internal rearrangement.In general, the treatments produced an increase in the gelatinization temperature andthey increased the thermo-stability below 95°C. Determinations of boiling-stable RSshowed an increase of its content in starches treated samples of corn, potato, and wheatin relation to their corresponding native starch, except for the cassava starch, which hada decrease of the content of RS. The corn starch treated during 60 minutes at 120ºCpresented the highest content in RS (4.2% w/w), followed by potato starch (3.1% w/w)with identical treatment. Although the increase of RS in corn and potato starches isimportant in relation to its corresponding native starch, the increase of RS in the sameones is not as important as we could expect; mainly if it is compared with other kinds ofstarches, for example with the corn starch of high native amylose and hydrothermallytreated.

ACKNOWLEDGMENTS

This work was partially supported by Fundación para el Desarrollo e Investigación Científica yTecnológica (DINCYT).

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Quantification of Resistant Starch in Several Starch Sources Treated Thermally

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