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International Journal of Food Sciences and Nutrition
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Studies on effect of multiple heating/cooling cycles on the resistant starch
formation in cereals, legumes and tubers
Baljeet S. Yadav a; Alka Sharma b; Ritika B. Yadav a
a Department of Food Science & Technology, Ch. Devi Lal University, Sirsa, Haryana, India b Department of
Food Technology, Guru Jambheshwar University of Science & Technology, Hisar, Haryana, India
Online Publication Date: 01 January 2009
To cite this Article Yadav, Baljeet S., Sharma, Alka and Yadav, Ritika B.(2009)'Studies on effect of multiple heating/cooling cycles on
the resistant starch formation in cereals, legumes and tubers',International Journal of Food Sciences and Nutrition,60:1,258 — 272
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Studies on effect of multiple heating/cooling cycles on
the resistant starch formation in cereals, legumes and
tubers
BALJEET S. YADAV
1
, ALKA SHARMA
2
& RITIKA B. YADAV
1
1
Department of Food Science & Technology, Ch. Devi Lal University, Sirsa, Haryana, India,
and
2
Department of Food Technology, Guru Jambheshwar University of Science & Technology,
Hisar, Haryana, India
Abstract
‘Resistant starch’ (RS) is defined as starch and starch degradation products that resist the action
of amylolytic enzymes. The effect of multiple heating/cooling treatments on the RS content of
legumes, cereals and tubers was studied. The mean RS contents of the freshly cooked legumes,
cereals and tubers (4.18%, 1.86% and 1.51% dry matter basis, respectively) increased to
8.16%, 3.25% and 2.51%, respectively, after three heating/cooling cycles (P50.05) with a
maximum increase of 114.8% in pea and a minimum of 62.1% in sweet potato (P50.05).
Significant positive correlations were observed between the RS content and amylose (y
0.443x5.993, r0.829, P50.05, n9) as well as between the percentage increase in RS
and insoluble dietary fiber content (y2.149x24.787, r0.962, P50.05, n9). A
differential scanning calorimeter study showed an increase in the T
0
,T
p
,T
c
and DHvalues of
the repeatedly autoclaved/cooled starches. The intact granular structure was also observed
disappear, as studied using scanning electron microscopy.
Keywords: Resistant starch, amylose, gelatinization, retrogradation, insoluble dietary fiber
Introduction
Starch occurs as insoluble and tightly packed granules in plant cells storing the
carbohydrates (Imberty et al. 1991). Besides being a major plant metabolite, starch is
an extremely important dietary component in the form of carbohydrates in the human
diet (Bjorck et al. 1994; Skrabanja et al. 1999). Recently, the research on starch has
been focused on its peculiar form, which is indigestible in vitro and in vivo (i.e.
resistant starch [RS]) (Sievert and Pomeranz 1989; Siljestrom et al. 1989; Gormley
and Walshe 1999; Tharanathan and Tharanathan 2001; Hoover and Zhou 2003;
Brouns et al. 2007; Englyst et al. 2007; Cummings and Stephen 2007; Sharma et al.
2008; Murphy et al. 2008; Grabitske and Slavin 2009). The digestion of starch is
mainly mediated by alpha-amylases (a-1,4-glucan hydrolase [EC3.2.1.1]), which act
on both amylose and amylopectin in an endo fashion releasing glucose, maltose,
oligosaccharides and higher dextrins into the lumen of small intestine. The glucose is
absorbed directly through the intestinal mucosa, whereas the oligosaccharides and
Correspondence: Dr Baljeet Singh Yadav, Lecturer, Department of Food Science & Technology, Ch. Devi
Lal University, Sirsa, Haryana 125055, India. E-mail: baljeet.y@rediffmail.com
ISSN 0963-7486 print/ISSN 1465-3478 online #2009 Informa UK Ltd
DOI: 10.1080/09637480902970975
International Jour nal of Food Sciences and Nutrition,
September 2009; 60(S4): 258272
Downloaded By: [Yadav, Baljeet S.] At: 03:47 9 October 2009
other dextrins are acted upon by the membrane bound glucosidases. These enzymes
include glucoamylase that cleaves a-1,4-glucan links from the non-reducing ends.
However, an increasing volume of evidence suggests that, with very few exceptions,
only a proportion of total ingested nutrients in a diet or in food is available, and the
term ‘availability’ has come into use. During food processing, derivatization of
nutrients and formation of cross-linkages occur, making the food inaccessible for
digestion and/or metabolism.
Englyst et al.’s (1982) studies on measurement of non-starch polysaccharides first
recognized the presence of a starch fraction resistant to enzymic hydrolysis. The term
‘resistant starch’ was first used to describe the incomplete digestion of starch in vitro that
had been cooked and cooled (Berry 1986). Now this term includes all starch and starch
degradation products that resist small intestinal digestion and enter the large bowel in
normal humans (Asp 1992). RS has been categorized into four types: RS
1
,RS
2
,RS
3
,
and RS
4
.RS
1
represents tightly bound starch molecules that are physically inaccessible
to digestive enzymes as these are wrapped in a fiber shell (Bird et al. 2000; Haralampu
2000) and it is found in partly milled grains and seeds. RS
2
is ungelatinized starch
granule, which is inaccessible to amylolytic enzymes due to its compact and unhydrated
structure. RS
3
is the retrograded or recrystallized starch and is found in most of the heat-
processed foods (Muir ad O’ Dea 1992; Haralampu 2000). RS
4
is the chemically
modified form; for example, esterified and cross-bonded starches that cannot be broken
down, since the modification process renders the structure inaccessible to digestion by
alpha-amylase (Cummings et al. 1996; Haralampu 2002).
RS is being examined both for its potential health benefits as well as functional
properties to produce high-quality foods. The beneficial short-chain fatty acids,
including mainly butyrate, acetate and propionate produced from the fermentation of
RS in the colon, may play an important role in the prevention of gastrointestinal
disorders including colon cancer, diverticulitis and hemorrhoids (Bird et al. 2000). RS
has been reported to lower the glycemic values, decrease serum cholesterol and
triglyceride levels, and increase fecal bulk and prebiotic effects (Nugent 2005; Sajilata
et al. 2006; Sharma et al. 2008).
Very little uncooked raw starch is consumed in normal diets and most of the
processed foods invariably involve the application of heat and moisture for varying
periods. During processing, the starch molecules undergo several physical modifica-
tions depending upon the type of starch and severity of the conditions applied (Goni
et al. 1996), leading to the formation of resistant starch. The amylase RS (RS
3
)is
formed in foods processed under relatively high moisture contents with cooking,
baking or autoclaving (Sagum and Arcot 2000; Tharanathan and Tharanathan 2001;
Habana et al. 2004; Katyal et al. 2005; Mahmood et al. 2006). Bravo et al. (1998)
studied the effect of different processing treatments on RS formation in moth beans,
horse gram and black gram. Vasanthan and Bhatty (1998) used annealing as a means
of increasing the RS content of pea and lentil starches from 8.4% to 14.1% and from
6.5% to 9.5%, respectively. Increases in the baking temperature and baking time were
found to result in increases in the RS contents of bakery products (Kale et al. 2002).
Three heating and cooling cycles resulted in increase in the RS content of potato
(Gormley and Walshe 1999). Sievert and Pomeranz (1989) described the use of
autoclaving/cooling cycles to produce RS from high-amylose maize varieties. Elevated
processing temperatures such as those used in canning enhance the effect of heating
and cooling. Canning/autoclaving has a greater depressive effect on the digestibility of
Multiple heating/cooling cycles and resistant starch formation 259
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starch and it is only partially ameliorated by reheating. It is important to study the
effect of multiple heating and cooling as foods such as potatoes and legumes may be
subjected to more than one cook/cool sequence as component of ready meals.
Although there have been different studies on RS formation during heating/cooling
treatments in food crops having high amylose content, like amylomaize, high amylose
barley, and so forth, very little and raw information is available on the effects of such
processing treatments on the RS formation in normally consumed food crops*
particularly in India. In view of the above, the present study aims to evaluate the
effect of multiple heating and cooling cycles on the RS content of some commonly
consumed cereals, legumes and tubers.
Materials and methods
Materials
Seeds of bengal gram or chickpea (Cicer arietinum), pea (Pisum sativum), lentil (Lens
esculenta), kidney beans (Phaseolus vulgaris), grains of wheat (Triticum aestivum), rice
(Oryza sativa), barley (Hordeum vulgare) and tubers of potato (Solanum tubersum) and
sweet potato (Ipomea batatas) were procured from the local market. The legume crops
were milled to dhals with the help of the locally available dhal mill. The flours (60
mesh particle size) from various legume and cereal crops were prepared using
Navdeep flourmill.
All chemicals used were of analytical grade. The enzymes used for analytical purpose
were pepsin (No. 7190, 2002 FIP U/G; Merck, Darmstadt, Germany), pancreatic
alpha-amylase (A-3176; Sigma, St Louis, MO, USA), amyloglucosidase from Aspergil-
lus niger (No. 10115; Fluka, Buchs, Switzerland), glucose oxidase (SRL 074040; SRL,
Mumbai, India) from A. niger, peroxidase from horseradish (RM 664; Himedia,
Mumbai, India), heat-stable alpha-amylase (No. A 3306; Sigma Chemicals, St Louis,
MO, USA) and protease enzyme (No. P 3910; Sigma Chemicals, St Louis, MO, USA).
Methods
The raw materials were analyzed for their moisture, ash, fat and protein contents by
employing the standard methods of analysis (AOAC 1984). The amylose content was
determined using the rapid colorimetric method of Williams et al. (1970). The dietary
fiber contents were determined by the AOAC enzymatic-gravimetric method (AOAC
2000), except that no corrections were made for protein and ash in the undigested
residue. The total starch content was determined as the glucose released by the
enzymic hydrolysis after gelatinization of the samples in boiling water (Goni et al.
1997). Rice starch was isolated by alkali steeping method of Wang and Wang (2001).
The starch from potato was extracted using the method of Peshin (2001). Bengal
gram starch was isolated by the method of Abia et al. (1993).
Multiple heating/cooling of the foods
The effect of multiple heating/cooling was studied on pressure-cooked legume flours,
cereal flours and tubers (15 psi, 1218C for 15 min; sample to water ratio of 1:5 for
cereal flours, 1:3 for legume flours and 1:2 for tubers). Cooling of the samples was
done at 48C for 24 h and reheating was done over a boiling water bath for 10 min after
260 B. S. Yadav et al.
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bringing the samples to the room temperature. Up to three heating/cooling cycles were
given to the samples.
Determination of resistant starch content
The RS was determined using the enzymic method of Goni et al. (1996). One
hundred milligrams of dry-milled sample or 400500 mg wet homogenized sample
was weighed in a 50 ml centrifuge tube. Pepsin solution (0.2 ml, 1 g pepsin/10 ml
KClHCl buffer, pH 1.5) was added to deproteinize the sample (408C for 60 min).
The cooled sample was treated with pancreatic alpha-amylase (A-3176; Sigma) (1.0
ml, 40 mg alpha-amylase per 1 ml Tris maleate buffer, 378C for 16 h) to hydrolyze
digestible starch. The pellet obtained after centrifugation (15 min, 3,000g) was
washed with distilled water and centrifuged again to discard supernatant. The pellet
was dispersed with 3.0 ml distilled water and 3.0 ml of 4 M KOH and mixed well with
a magnetic stirrer along with constant shaking for 30 min at room temperature. After
the complete dispersion of sample, 5.5 ml of 2 M HCl and 3 ml of 0.4 M sodium
acetate buffer (pH 4.75, pH adjusted with 2 M HCl) and 80 ml amyloglucosidase
(5 mg/ml acetate buffer pH 4.75) were added and the sample placed in a water bath at
608C for 45 min with constant shaking. The contents were centrifuged (15 min,
3,000g), and supernatant collected in a 500 ml volumetric flask. The residue was
washed with 10 ml distilled water, centrifuged again and the supernatant was
combined with previously obtained one. The volume was made to 250500 ml
depending upon the RS content. The amount of glucose was determined using
glucose oxidaseperoxidase reagent.
RS (percentage of the sample as is) was calculated as:
% RS contentglucose concentration from standard curve 0:9
volume correctionx 1=1;000 100=w
The% RS content was calculated on a dry matter basis.
Resistant starch preparation from native starches
RS formation was studied using differential scanning calorimetery and scanning
electron microscopy in the repeatedly autoclaved/cooled starches isolated from rice,
bengal gram and potato. The isolated starch (25g, as is basis) of each crop was
suspended with distilled water with a starch/water ratio of 1:3.5 in a 500 ml beaker.
The suspensions were autoclaved in a thermostatically controlled autoclave at 128C
for 1 h. After autoclaving, the samples were allowed to cool at room temperature and
stored overnight in a refrigerator at 48C. The autoclaving/cooling cycles were repeated
five times and the treated samples were wet-milled in a pestle and mortar, and dried in
a vacuum oven at 558C. The dried material was ground in a blender and allowed to
pass through a sieve with 250 mm openings.
Differential scanning calorimetry
The thermal properties of isolated native and repeatedly heated/cooled starches
isolated from bengal gram, rice and potato were analyzed using a differential scanning
calorimeter (DSC-821; Mettler Toledo, Schwerzenbach, Switzerland) equipped with
a thermal analysis data station. Starch (10 mg, dry matter basis) was weighed in
Multiple heating/cooling cycles and resistant starch formation 261
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aluminum pan (ME 27331; Mettler Toledo). About 20 ml distilled water was added
with the help of a Hamilton microsyringe to achieve a starch-water suspension
(Szczodrak and Pomeranz 1991). The pans were sealed hermetically and allowed to
stand for 1 h at room temperature before heating in the differential scanning
calorimeter. A pan with water served as reference. The samples were heated at the rate
of 108C/min from 20 to 1808C.
Scanning electron microscopy
Scanning electron micrograms of native and repeatedly heated/cooled starches
isolated from bengal gram, rice and potato were obtained with a scanning electron
microscope (Jeol JSM-6100; Jeol Ltd, Tokyo, Japan). For examination by scanning
electron microscopy, finely ground and ethanol dehydrated samples were placed on an
aluminum stub and the samples were coated with a thin film of gold, with the help of
the Jeol ion sputter (JFC-1100). An acceleration potential of 10 kVA was used during
micrography.
Statistical analysis
A randomized complete block design with three replications was used to analyze changes
in RS content during multiple heating/cooling cycles. Data were analyzed using two-way
analysis of variance procedures. Statistical analysis was performed using the OPSTAT
software version opstat1.exe (Hisar, India). Correlation studies were performed using
SPSS 11.0 software (SPPS 11.0 Chicago, Illinois).
Results and discussion
Starch and dietary fiber profile of food crops
Among legumes, bengal gram showed the highest value of total starch content
(percentage dry matter basis); that is, 60.26%. The values of the total starch content
for pea and chickpea were comparable with those found by Rosin et al. (2002). The
amylose content of legumes was higher than that of cereals and tubers in general. The
amylose content of legumes was in agreement with those observed by Rosin et al.
(2002). The total starch content of different cereal food crops varied from 65.54% for
barley to 81.42% for rice. Among cereals, the amylose content calculated as the
percentage of total starch was the highest for wheat (25.85%) and lowest for rice
(20.66%). The total dietary fiber (TDF) values of legumes varied from 17.18%
(percentage dry matter basis) for kidney beans to the highest value of 24.92% for
bengal gram. Bravo et al. (1999) also observed comparable value of TDF for bengal
gram. The observed values of insoluble dietary fiber (IDF) and TDF for lentil,
common bean and pea in this study were in agreement with the values observed by
Almeida Costa et al. (2006). Among cereals, barley was found to show the highest
value of TDF; that is, 15.02%.
Resistant starch content of repeatedly heated/cooled foods
The RS content of the freshly cooked legumes was the highest, followed by cereals and
tubers in general. The RS content of rice was the minimum and was 1.24%, compared
262 B. S. Yadav et al.
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with a maximum RS content of 4.89% in freshly cooked flour of kidney beans (Table
I). In general, legume starches differ from cereal and tuber starches both in their
chemical composition and granular structure (Doublier 1987; Hoover and Zhou
2003). The reduced bio-availability of starch and hence the greater RS content of
legumes can be attributed to the presence of intact tissue/cell structures enclosing
starch granules, a high level of amylose (2233% in this study), a high content of
viscous soluble dietary fiber components, the presence of large number of antinu-
trients, ‘B’-type crystallites and strong interactions between amylose chains (Wursch
et al. 1986; Siddhuraju and Becker 2001; Mahadevamma et al. 2003). Permanence of
intact starch granules trapped within the cells in the pre-cooked legume flour was
observed by Tovar et al. (1991) even after extensive homogenization and pepsin
treatment. This could account for the higher RS content in the freshly cooked
legumes. The lower RS content of cereals could be expected from their polymorph
starch type. Cereal starches are ‘A’-type starches (Gernat et al. 1990; Cairns et al.
1997) always having a low RS fraction of starch. The tubers such as potato and sweet
potato in their natural state contain sufficient water to allow full gelatinization of their
starch content during heat treatment as demonstrated by their low RS content. The
rapid expansion of tuber starch as it gelatinizes in the presence of excess water loosens
the cellular architecture of most potato products, which allows easy access for starch-
degrading enzymes.
Multiple heating/cooling greatly increased the RS content of foods (Table I). The
mean RS content of the freshly boiled food crops was 2.81% and the mean value of RS
Table I. RS content (percentage dry matter basis) of repeatedly heated/cooled foods.
Food crop Control
a
Once-heated/
cooled
Twice-heated/
cooled
Thrice-heated/
cooled
Mean
Wheat 1.7690.14
B
2.4990.14
C
(41.4)
2.8790.18
C
(63.0)
3.3190.10
C
(88.0)
2.61
C
Rice 1.2490.09
A
1.6290.09
A
(30.6)
1.8890.12
A
(51.6)
2.1290.08
A
(70.9)
1.71
A
Barley 2.5790.09
C
3.5290.22
D
(36.9)
4.1190.14
D
(59.9)
4.3190.12
D
(67.7)
3.63
D
Bengal gram 4.5590.20
F
7.0990.17
G
(55.8)
8.6390.14
H
(89.6)
9.1990.08
G
(101.9)
7.36
G
Pea 3.1690.11
D
5.369.0.13
E
(69.6)
5.9390.11
E
(87.6)
6.7990.18
E
(114.8)
5.31
E
Lentil 4.8990.27
F
6.4890.07
F
(32.5)
8.1390.10
G
(66.2)
9.2190.08
G
(88.3)
7.18
G
Kidney bean 4.1290.08
E
5.3290.13
E
(29.4)
6.5390.15
F
(58.8)
7.4490.14
F
(81.0)
5.85
F
Potato 1.7090.08
B
2.1090.05
B
(23.5)
2.4290.09
B
(42.3)
2.8890.05
B
(69.4)
2.28
B
Sweet
potato
1.3290.16
A
1.5990.16
A
(20.4)
1.9390.05
A
(46.2)
2.1490.03
A
(62.1)
1.75
A
Mean 2.81
A
3.95
B
4.71
C
5.27
D
Data presented as mean9standard deviation of three independent determinations at 5% level of
significance (PB0.05). Values in parentheses show the percentage increase over the control value. Values
with different uppercase superscript letters in the column (mean values) differ significantly.
a
Freshly
autoclaved.
Multiple heating/cooling cycles and resistant starch formation 263
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in the thrice-heated/cooled foods was observed to be 5.27%, indicating a significant
increase of about 87.5% (P50.05). The RS content of the once-heated, twice-heated
and thrice-heated/cooled foods was significantly different (P50.05). A significant
interaction (P50.05) was also observed between heating/cooling treatments and
crops; that is, the effect of three successive heating/cooling treatments on the RS
content was more pronounced in legumes as compared with cereals and tubers. A
maximum increase of 114.0% in the RS content was observed in case of thrice-heated/
cooled pea flour. Among thrice-heated/cooled food crops, sweet potato reported a
minimum increase of 62.1% in RS content. Although rice showed a higher increase in
the RS content as compared with sweet potato at every stage of heating/cooling, no
significant difference was observed in the RS content of rice and sweet potato at any
stage of heating/cooling (PB0.05).
Szczodrak and Pomeranz (1991) observed the formation of RS in high amylose
barley starch with increasing number of autoclaving/cooling cycles. The percentage
yield of RS was 5.8 in once-heated/cooled barley starch and it increased to 25.8 after
20 heating/cooling cycles. However, the percentage RS content of repeatedly heated/
cooled barley flour observed in the present study was less because of low amylose
content of the barley cultivar used and the lesser number of heating/cooling cycles,
resulting in formation of lesser retrograded starch. Sievert and Pomeranz (1989) also
observed a pronounced effect of the number of heating/cooling cycles on the RS
content of wheat, maize, potato, pea, waxy maize and amylomaize. The starch/water
ratio and autoclaving temperature affected the formation of RS in amylomaize VII
starch also. Gruchala and Pomeranz (1993) reported an increased yield of RS in
repeatedly autoclaved/cooled amylomaize starch. A consistent increase in the yield of
RS content was observed up to four autoclaving/cooling cycles, and it increased from
20.7% after one heating/cooling cycle to 31.7% after four cycles. RS was also found to
develop and increase in bengal gram and red gram dhals autoclaved/cooled for four
cycles (Mahadevamma et al. 2003). A similar increase in the RS content has been
observed in multiple heated/cooled potatoes by Kingmann and Englyst (1994). The
findings of the present study are also in agreement with the observations reported by
Gormley and Walshe (1999) in multiple cooked/cooled potatoes.
The percentage increase in the RS content of repeatedly heated/cooled tubers was
less in comparison with that of legumes and cereals. The two possible reasons for this
may be the low amylose content in tuber crops as amylose plays an important role in
the formation and development of RS during heating/cooling treatments and,
secondly, the tuber crops used in this study were not in the form of flours as in the
case of cereals and legumes. In the case of flours the starch is gelatinized more
uniformly and intensively, and hence is retrograded more as compared with intact
grains or tubers.
RS is formed during retrogradation or recrystallization of the gelatinized starch,
particularly amylose. Each successive cooking increases the degree of starch gelatiniza-
tion and each cooling promotes more retrogradation. The gelatinized starch recrys-
tallizes into a more ordered solid state that is less susceptible to the action of pancreatic
amylase. Most of the crystallized starch chains are re-dispersed by reheating, leading to
restoration of digestibility, but a small fraction of mainly retrograded amylose (RS
3
)
remains resistant (Englyst et al. 1982). During each reheating and cooling cycle a bit
more RS
3
is formed, leading to an increased percentage of RS content each time in
repeatedly heated/cooled foods. Autoclaving is more effective in increasing the RS
264 B. S. Yadav et al.
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content of most of the starches, presumably because it mobilizes the starch polymers by
swelling of the native granules ultrastructure and thereby allowing separation of the
amylose domain that crystallizes more readily upon cooling (Russell et al. 1989). A
positive and significant correlation (y0.443x5.993, r0.829, P50.05, n9) was
observed between the RS of repeatedly heated/cooled foods and amylose. Figure 1
depicts the RS content of repeatedly heated/cooled foods as affected by amylose. Sagum
and Arcot (2000) also observed a higher amount of RS for mation in the pressure-cooked
rice cultivars having higher amylose content (1.6% RS with 20% amylose and 2.8% RS
with 31% amylose). Rosin et al. (2002) also observed a positive significant correlation
between the RS of cooked foods and amylose (r0.631). The percentage increase in the
IDF of repeatedly heated/cooled foods (Figure 2) was also observed to relate positively
with the amylose content (y2.149x24.787, r0.962, P50.05, n9), suggesting
that retrogradation of amylose occurred in repeatedly heated/cooled foods, resulting in
enhanced levels of RS content. A positive significant correlation (r0.81) between IDF
and RS content was also observed by Thed and Phillpps (1995) in potatoes.
0
1
2
3
4
5
6
7
8
9
10
0 10203040
Am
y
lose content (% starch)
%RS (dmb)
% RS
Linear (% RS)
Figure 1. RS content of repeatedly heated/cooled foods as affected by amylose content (r0.82). dmb, dry
matter basis.
0
10
20
30
40
50
60
0 50 100 150
%Increase in RS content
% Increase in IDF content
%Increase in IDF
Linear (%Increase in IDF)
Figure 2. Percentage increase in IDF content versus percentage increase in the RS content of repeatedly
heat-treated and cooled food crops (r0.91).
Multiple heating/cooling cycles and resistant starch formation 265
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Differential scanning calorimetric study
The gelatinization transition temperatures T
0
(onset), T
p
(peak), T
c
(completion) and
enthalpy of gelatinization (DH) of native and heated/cooled starches of bengal gram,
potato and rice are presented in Table II. The values of transition temperatures (T
p
)
for the native starches were found to be lower than the normal expected values, which
may be due to difference in the starchwater ratios or experimental set-up. The
difference in the transition temperatures of the different starches suggests that
the starches may differ with respect to their crystallite size and/or the degree of
crystallite association within the granule. The differences in DHamong different
starches probably reflect differences in the number of double helices (within
amorphous and crystalline regions of granule) unraveling and melting during
gelatinization (Levine and Slade 1988). Cooke and Gidley (1992) have also shown
by X-ray spectroscopy that DHvalues reflect mainly the loss of double-helical order
rather than crystalline regions. An increase in the T
0
,T
p
,T
c
and DHover that of native
starches was observed in multiple autoclaved/cooled starches. Mangala et al. (1999)
observed a similar increase in the transition temperatures and enthalpy values in the
autoclaved and cooled starches of rice and ragi. T
p
is an indication of structural
stability and resistance to gelatinization. The crystallinity of the heated/cooled starches
is governed by the cooling in between the heating steps. Autoclaved and cooled
starches develop more stabilized structures with increased degree of ordering. The
thermograms of native and repeatedly heated/cooled starches of potato, bengal gram
and rice are shown in Figure 3. The melting enthalpy values for native starches varied
from 2.65 to 3.29 J/g, whereas for autoclaved/cooled starches the values varied from
7.99 to 12.79 J/g. The higher melting enthalpies of heated/cooled starches might be
related to the stabilization of the starch molecules through retrogradation and
compact structures formation. The thermograms of amylomaize preparations also
showed that an increase in the number of autoclaving/cooling cycles was associated
with an increase in melting enthalpies. The corresponding RS residue also exhibited
sharper endotherms over a broader temperature range [Sievert and Pomeranz 1989].
Scanning electron microscopy
The structural differences between the native and heat-treated starches were
illustrated using scanning electron microscopy. Native starches of potato, rice and
Table II. Thermal properties of the native and repeatedly heated/cooled starches.
Transition temperature (8C) DH(J/g)
Starch T
0
T
p
T
c
Bengal gram 45.37 56.96 68.36 3.29
Bengal gram
a
64.25 68.89 73.46 7.99
Potato 48.21 58.99 70.77 2.65
Potato
a
60.20 69.12 80.6 7.62
Rice 55.11 63.45 74.70 2.66
Rice
a
56.69 60.85 69.37 12.79
T
0
Temperature at which gelatinization of starch starts; T
p
Tis temperatures measures the thermal
stability of starch; T
c
The temperature at which gelatinization concludes; DHenthalpy associated with
gelatinization process.
266 B. S. Yadav et al.
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bengal gram consist of granules of varying diameter. Figures 4 and 5 respectively show
the structural features of native and repeatedly heated/cooled starches. The starches
showed completely different images after repeated autoclaving/cooling treatment. The
intact granular structure disappeared and bigger irregularly shaped particles with a
continuous sponge like porous network with some compact structures appeared. The
Figure 3. Differential scanning calorimetery endotherms of (line A) native and (line B) repeatedly heated/
cooled (I) potato, (II) bengal gram and (III) rice starch.
Multiple heating/cooling cycles and resistant starch formation 267
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higher melting enthalpies of repeatedly heated/cooled starches might be related to this
stabilization. This transformation of the structure of the starch granules upon heating
and cooling make them resistant to amylolytic attack, and it is generally also
acknowledged that alpha-amylase preferentially attacks amorphous regions of starch
and that solid regions of starch are hydrolyzed at a slower rate (Vasanthan and Bhatty
1998). The findings of the present study are in agreement with those of Sievert and
Pomeranz (1989).
Figure 4. Scanning electron micrographs of (a) native rice starch, (b) bengal gram starch and (c) potato
starch.
268 B. S. Yadav et al.
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Conclusion
From the results it has been concluded that repeated heating/cooling of foods results
in an increase in their RS content. Although a linear correlation (r0.829) between
amylose content and RS content has been observed, the increasing RS is not
proportional to the increasing amylose content. It implies that along with amylose
content there are some other factors that also influence the RS content, and these may
Figure 5. Scanning electron micrographs of (a) repeatedly heated/cooled rice starch, (b) bengal gram starch
and (c) potato starch.
Multiple heating/cooling cycles and resistant starch formation 269
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include amylose chain lengths, granule size, type of crystalline polymorphs, physical
insulation of starch by thick-walled cells, porosity and physical distribution of starch in
relation to the dietary fiber components, and so forth. In fact, RS formation is a very
complex process that involves several parameters simultaneously which must be
properly quantified in foods. Similarly, an increase in RS content is also not strictly
proportional to the increasing IDF content. This also once again signifies that the
enzymic-gravimetric method for the estimation of dietary fiber is not reliably inclusive
of RS in the IDF fraction. It is therefore significant to redefine dietary fiber. However,
it could act as an incentive for food manufacturers in raising levels of RS in processed
foods as a means of generating extra ‘dietary fiber’. It is important to study such
effects since foods are often cooked and held chilled, as in potato-salad manufacture.
In distilleries and breweries, such studies may be of great importance as cooking and
cooling operations are repeated and there occurs formation of RS in the residue if
cooker vessels are not emptied or cleaned properly. The RS formed may cause
problems of filtration and haze formation in beer. It is important to emphasize here
that RS should be included in the food composition database as it plays important
physiological roles in the body.
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