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Resistant star Starch Starch is widely distri ingredient of many foods, it nutrition(1). In plants, starch and tightly packed manner. C glucopyranosyl units linked to Starch is composed o residues linked with α-D-(1branched molecule with α-D-( polymerization of 100–10,000 Amylopectin isthe major com 30% of the starch. Pure wax varieties of maize and rice hav rch in foods and its health be ibuted in various plant organs as a storage c t is also the most important carbohydrate h occurs as granules, storing the carbohydra Chemically, starches are polysaccharides com ogether with α-D-(1–4) or α D-(1–6) linkages. of amylose, which is essentially a linear pol 1–4) linkages (Fig. 1), and amylopectin w (1–4) and α-D-(1–6) linkages (Fig. 2). Amylo 0 DP, whereas, amylopectin has an average mponent of starch, whereas, amylose typica xy starches are almost 100% amylopectin ve 30–70% amylose.(18) Fig. 1 Structure of amylose Fig. 2 Structure of amylopectin 1 enefits arbohydrate. As an e source in human ates in an insoluble mposed of alpha-D- .(18) lymer with glucose which is the larger ose has a degree of e DP of 2 millions. ally constitutes 15and high amylose

Resistant Starch

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Page 1: Resistant Starch

Resistant starch

Starch

Starch is widely distributed in various plant organs as a storage carbohydrate. As an

ingredient of many foods, it is also the most important carbohydrate source in human

nutrition(1). In plants, starch occurs as granules, storing the carbohydrates in an insoluble

and tightly packed manner. Chemically, starches are polysaccharides composed of alpha

glucopyranosyl units linked together with α

Starch is composed of amylos

residues linked with α-D-(1–

branched molecule with α-D-(1

polymerization of 100–10,000 DP, whereas, amylopectin has an average DP of 2 millions.

Amylopectin isthe major component of starch, whereas, amylose typically constitutes 15

30% of the starch. Pure waxy starches are almost 100% amylopectin and high amylose

varieties of maize and rice have 30

tarch in foods and its health benefits

Starch is widely distributed in various plant organs as a storage carbohydrate. As an

ingredient of many foods, it is also the most important carbohydrate source in human

. In plants, starch occurs as granules, storing the carbohydrates in an insoluble

and tightly packed manner. Chemically, starches are polysaccharides composed of alpha

anosyl units linked together with α-D-(1–4) or α D-(1–6) linkages.

Starch is composed of amylose, which is essentially a linear polymer with glucose

(1–4) linkages (Fig. 1), and amylopectin which is the larger

(1–4) and α-D-(1–6) linkages (Fig. 2). Amylose has a degree of

00 DP, whereas, amylopectin has an average DP of 2 millions.

Amylopectin isthe major component of starch, whereas, amylose typically constitutes 15

30% of the starch. Pure waxy starches are almost 100% amylopectin and high amylose

ce have 30–70% amylose.(18)

Fig. 1 Structure of amylose

Fig. 2 Structure of amylopectin

1

in foods and its health benefits

Starch is widely distributed in various plant organs as a storage carbohydrate. As an

ingredient of many foods, it is also the most important carbohydrate source in human

. In plants, starch occurs as granules, storing the carbohydrates in an insoluble

and tightly packed manner. Chemically, starches are polysaccharides composed of alpha-D-

6) linkages.(18)

e, which is essentially a linear polymer with glucose

, and amylopectin which is the larger

. Amylose has a degree of

00 DP, whereas, amylopectin has an average DP of 2 millions.

Amylopectin isthe major component of starch, whereas, amylose typically constitutes 15–

30% of the starch. Pure waxy starches are almost 100% amylopectin and high amylose

Page 2: Resistant Starch

In the native form of starch, amylose and amylopectin molecules are organised in

granules as alternating semi-

semi-crystalline layer consists of of double helices formed by short amylopectin b

most of which are further ordered

lamellae. The amorphous regions of the semi

composed of amylose and non

Fig. 3 Structure of a starch granule, with alternating amorphous and semi

zones constituting the growth rings

Starch can be categorized in to 3 groups by

1) Type A has amylopectin chain lengths of 23 to 29 glucose units formed as a

double helix with amylose moieties packed inside, common in cereals.

2) Type B structure consists of amylopectin of chain lengths of 30 to 44 glucose

units with water interspersed and

3) Type C appears to be a mixture of both A and B forms as found in legumes

In the native form of starch, amylose and amylopectin molecules are organised in

-crystalline and amorphous layers that form growth rings.

crystalline layer consists of of double helices formed by short amylopectin b

most of which are further ordered into crystalline structures known as the crystalline

lamellae. The amorphous regions of the semi-crystalline layers and the amorphous layers are

composed of amylose and non-ordered amylopectin branches (Fig. 3). (11)

tructure of a starch granule, with alternating amorphous and semi

zones constituting the growth rings (11)

Starch can be categorized in to 3 groups by X-ray diffraction pattern (Fig. 4):

Type A has amylopectin chain lengths of 23 to 29 glucose units formed as a

double helix with amylose moieties packed inside, common in cereals.

Type B structure consists of amylopectin of chain lengths of 30 to 44 glucose

units with water interspersed and is usually found in raw potato and green banana starch.

Type C appears to be a mixture of both A and B forms as found in legumes

Fig. 4 X-ray diffraction

diagrams of A-

type starch (1)

2

In the native form of starch, amylose and amylopectin molecules are organised in

crystalline and amorphous layers that form growth rings. The

crystalline layer consists of of double helices formed by short amylopectin branches,

into crystalline structures known as the crystalline

crystalline layers and the amorphous layers are

(11)

tructure of a starch granule, with alternating amorphous and semi-crystalline

pattern (Fig. 4):

Type A has amylopectin chain lengths of 23 to 29 glucose units formed as a

Type B structure consists of amylopectin of chain lengths of 30 to 44 glucose

is usually found in raw potato and green banana starch.

Type C appears to be a mixture of both A and B forms as found in legumes.

ray diffraction

-type and B-

Page 3: Resistant Starch

3

Starch digestion

The digestion of starch is mediated by salivary and pancreatic α- amylases that release

glucose, maltose, oligosaccharides, and higher dextrins into the lumen of small intestine.(18)

The remaining hydrolysis takes place by the action of enzymes located in the brush border of

the intestinal mucosa. Only monosaccharides can enter the mucosal cell. Glucoamylase

(maltase) hydrolyzes maltose and the straight chain oligosaccharides to glucose. Sucrose is

hydrolyzed by sucrase to fructose and glucose. Lactose is similarly hydrolyzed by lactase

(beta-galactosidase) to glucose and galactose. The limit dextrins are hydrolyzed to glucose by

alpha-(1,6)-glucosidase.(22)

Resistant starch

Before the early 1980s, starch was assumed to be fully digestible in human intestine.

In 1982, Englyst et al. first recognized the presence of starch fraction resistant to enzymic

hydrolysis during their research on measurement of nonstarch polysaccharides. The name

“Resistant starch” was defined as all starch and starch degradation products that resist small

intestinal digestion and enter large bowel in normal humans.(18)

Enzymatic resistance of starch may affect by several intrinsic or extrinsic factors.

Intrinsic factor included food particle size, amylose-lipid complex, enzyme inhibitors, starch

granule structure, amylose/amylopectin ratio. Extrinsic factor included different processing

treatments to foods.(18)

Depending on the various reasons for the enzyme resistance, the resistant starch can

be categorized into four groups:(18)

RS1 (physically inaccessible starch) represents the starch granules which enclosed in

the intact cell walls and inaccessible to the digestive enzymes. It is found in partly milled

grains and seeds. This type of starch is heat stable in normal cooking operations. But can be

totally digested if properly milled.

RS2 (resistant starch granule) is raw, ungelatinized native starch molecule in granular

form with B-type crystallinity.

RS3 (retrograded starch) is mainly the retrograded amylose formed during cooling of

gelatinized starch. It can only be dispersed with KOH or dimethyl sulfoxide. It can be found

in the heat-processed foods such as cooked and cooled potatoes and breads. Starch

digestability can by improved by reheating.

RS4 (chemically modified starch) has cross-bonding with chemical reagents such as

ether and ester. This type of starch cannot be broken down by digestive enzyme since the

structure of the starch molecule is modified.

Measurement of resistant starch

Many methods were developed for measurement of resistant starch including Englyst

et al. (1982), Berry (1986), Englyst et al. (1992), Champ (1992), Muir and O'Dea (1992),

Faisant et al. (1995), Goñi et al. (1996), Akerberg et al. (1998) and Champ et al. (1999).

Page 4: Resistant Starch

4

McCleary and Monaghan (2002) developed a method for measurement of RS. Many

factors which affect RS result were studied included pepsin pretreatment, pH of incubation,

shaking or stirring, effect of maltose and inclusion of amyloglucosidase.

In the physiological digestive process, foods are subjected to protease (pepsin)

hydrolysis in stomach at about pH 2.0 before entering small intestine. The use of pepsin may

be need because in food high in protein such as beans, protein may partially encapsulate the

starch. However, the result (Table 1) demonstrated that protease pretreatment had no

insignificant effect on RS values. Because crude pancreatic α-amylase contains active

proteases (trypsin 50 mU/mg, chymotrypsin 325 mU/mg pancreatic α-amylase) which is

probably adequate for release starch from the protein matrix.

Table 1 Effect of pepsin pretreatment on determined RS content of sample

Sample type Pepsin pretreatment RS, % (w/w)

Batchelors kidney beans (canned, lyophilized) Yes 5.10 ± 0.1

No 5.00 ± 0.1

Rob Boy flageolet beans (canned, lyophilized) Yes 4.55 ± 0.05

No 4.40 ± 0.10

Native potato starch Yes 73.9 ± 0.06

No 75.6 ± 1.05

Green banana (lyophilized) Yes 50.8 ± 0.51

No 48.0 ± 0.20

Maltose occurred originally in foods or by activity of α-amylase can inhibit pancreatic

α-amylase by competitive inhibition, thus caused higher RS values (Table 2). After addition

of maltase, RS value were lower, indicated that pancreatic α-amylase was more active (Table

3).

Table 2 Effect of added maltose on determined RS values

Sample Added maltose, mg Determined RS, % (w/w)

Hi Maize 1043 0 45.4

50 51.2

CrystaLean 0 42.6

50 45.1

Potato amylose 0 41.2

50 43.6

Novelose 330 0 41.8

50 45.9

Potato starch 0 9.1

50 9.7

Regular maize starch 0 9.5

50 15.3

100 19.6

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5

Table 3 Effect of added maltase enzyme on the determined level of RS for RMS and HAMS

Sample RS, % *w/w)

No maltase added With maltase

RMS 6.2 0.8

HAMS (60107) 46.3 38.0

Maltase added at a level of 500 units/test

However, addition of amyloglucosidase (AMG) in incubation mixture resulted in

lower amount of RS than that added with maltase, indicated that AMG can also remove

inhibitory effect of maltose. In many in vitro method AMG is added to ensure complete

hydrolysis of soluble starch fragments and maltosaccharides to glucose. Using either pure or

crude pancreatic α-amylase in the presence of AMG, similar RS values were obtained,

confirmed that purity of RS was not an issue (Table 4).

Table 4 Effect of amyloglucosidase (AMG) and maltase on the determined level of RS in

HAMS

Pancreatic α-amylase AMG or maltase RS, % (w/w)

Pure (Sigma Cat. No. A-2643) Neither 58.2/59.6

Maltase (500 U) 47.6/49.4

AMG (12 U) 39.0/41.1

Crude (pancreatin) AMG (12 U) 42.2/43.4

The method of McCleary and Monaghan (2002) was adopted by Association of

Official Analytical Chemists (AOAC) as a first action of official method for measurement of

resistant starch in starch and plant materials by enzymatic method (Method 2002.02). The

method consists of 2 main steps – hydrolysis of non-resistant starch and measurement of RS.

Brief steps of the method were shown as follows:

1) Hydrolysis of non-resistant starch

Sample grinding

Pancreatic α-amylase + amyloglucosidase 16 h at 37°C

with shaking 200 stroke/min

Enzyme termination by ethanol or industrial methylated spirit

Centrifugation

Decant supernatant

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6

2) Measurement of RS

Starch pellet

Dissolve in 2M KOH by stirring in ice-water bath

Neutralize with acetate buffer

Hydrolyzed to glucose with amyloglucosidase

Measure glucose with glucose oxidase-

peroxidase reagent (GOPOD)

In addition, non-resistant starch (solubilized starch) can be measured by pooling

supernatant and measure glucose with GOPOD.

Results obtained by this method are in best agreement with currently available in vivo

data and other published in vitro data as shown in Table 5. Most methods used pancreatic α-

amylase with amyloglucosidase and a shaking-tube format.

Table 5 Comparison of RS values obtained using several in vitro analytical methods and in

vivo results

Source of starch

RS (in vitro method/results) RS

(in vivo

results) Englyst Faisant Champ McCleary Goni

Potato starch (native) 66.5 83.0 77.7 77.0 – 78.8

Amylomaize starch

(native)

71.4 72.2 52.8 51.7 – 50.3

Amylomaize starch

(retrograded)

30.5 36.4 29.6 42.0 37.8 30.1

Bean flakes 10.6 12.4 11.2 14.3 15.3 9-10.9

Corn flakes 3.9 4.9 4.3 4.0 4.7 3.1-5.0

Canned beans 17.1 – 17.1 16.5 – 16.5

ActiStar 63 – 57 58.0 57 59

Values are presented as a percentage of total starch content of sample.

From: McCleary and Monoghan (2002)

Resistant starch content in foods

Resistant starch content in foods varies by type and variety of raw materials, and

processing condition, as shown in Table 6. Raw bananas have high resistant starch, more than

50% by dry basis, and vary by varieties. Raw rice has RS range from 4 to 34. But RS in

cooked or processed rice were less than 10%. Studies of RS in legumes are quite extensive.

Most raw legumes have RS more than 10%, except chickpea and lentil. Soaking, cooking and

sprouting cause lower RS content. Among tubers, taro has high RS about 43%, sorghum has

RS about 5-6%. Bakery products have very low RS.

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7

Table 6 Resistant starch content in various foods

Food Starch content (% DB)

Method Ref. Total Resistant

Rice and rice products

Saohai cultivar (hard rice), raw 52.0 34.8 Goni (1996) (21)

Dookmali cultivar (soft rice), raw 67.5 11.6

Brown rice, raw 66.6 4.2

White rice, cooked 64.7 7.1

Khanomjean, cooked 41.9 8.5

Kaotung, fried 75.9 2.6

Kaokreupvor, roasted 54.6 2.9

Kaokreup, fried 44.9 2.0

Extruded rice snack 4.2 0.4

Noodle type products, uncooked

Glass noodles 59.0 11.3 Goni (1996) (21)

Instant glass noodles 60.7 9.1

White rice noodles 67.0 3.0

Brown rice noodles 73.7 2.2

Instant rice noodles 57.1 2.4

Vermicelli 58.5 4.4

Rice sheet 41.3 2.4

Spaghetti <1 (6)

Legumes

Mungbeans, raw 44.3 22.9 Goni (1996) (21)

Red kidney bean, raw 41.6 35.0 Englyst (1992) (5)

Red kidney bean, soaked and boiled 46.0 2.5

Black bean, raw 39.8 18.3 Goni (1996) (21)

Black bean, steam heated 38.7 6.0 Tovar (1990) (20)

Mothbean, raw 39.54 12.20 Goni (1996) (2)

Mothbean, cooked 46.24 3.90

Mothbean, water soaked and cooked 47.88 3.72

Mothbean, sprouted and cooked 44.75 2.67

Horse gram, raw 36.03 26.42

Horse gram, cooked 47.32 5.21

Horse gram, water soaked and cooked 46.29 5.69

Horse gram, sprouted and cooked 44.66 4.44

Black gram, raw 37.87 19.66

Black gram, cooked 40.73 3.40

Black gram, water soaked and cooked 41.02 3.68

Black gram, sprouted and cooked 38.99 3.03

Green pea, raw 53.4 32.1 Englyst (1992) (5)

Green pea, soaked and boiled 53.1 5.8

Yellow pea, raw 57.1 36.2

Yellow pea, soaked and boiled 63.9 10.3

Chickpea, raw 3.39 Faisant et al. (4)

Lentil, raw 3.25

Tubers

Taro 43.2 AACC (1995) (17)

Sweet potato 9.05

Cassava chip 52.4 Englyst (1992) (15)

Cassava pellet 40.9

Tapioca pearls (sago) 44.7 4.5 Goni (1996) (21)

Page 8: Resistant Starch

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Food Starch content (% DB)

Method Ref.

Total Resistant

Banana

Namwa 53.0 AACC (1995) (17)

Hom Thong 47.2

Banana flour (green 90-120 days) Goni (1996) (21)

Namwa 79.7 56.6

Hom 91.0 57.7

Kai 80.5 52.2

Lepmurnang 72.1 57.0

Hugmuk 72.3 61.4

Hin 72.7 68.1

Ngachaeng 75.5 64.6

Lepchaengkut 88.7 50.7

Nangphaya 91.0 66.8

Phamahagkuk 91.4 60.1

Thepparod 82.4 58.5

Seeds and grains

Sorghum 6.46 McCleary(2002) (10)

Sorghum (autoclaved 130°C) 5.24

Job’s tears 66.5 6.4 Goni (1996) (21)

Bakery products

Biscuit 1.8 Goni (1996) (6)

White bread (crumb) 2.3

Crispbread <1

Effects of processing on resistant starch content in foodstuffs

1. Heating

Cooking of presoaked legumes decrease RS level in pea (Pisum sativum L. cv.

Maria), common bean (Phaseolus vulgaris L. cv. IAC carioca Eté), chickpea (Cicer

arietinum L.) and lentil (Lens culinaris Med. cv. Silvina). The grains of the legumes were

chosen to eliminate external material, immature seeds and damaged grains. Part of the grains

of each legume was ground raw into flour. The rest were washed in running water, soaked for

a period of 16 h (1:2w/v) and then cooked with the addition of one volume of water.

Common bean and chickpea grains were cooked in a pressure cooker (14.7 psi) for 20 and 40

min, respectively. Pea and lentil legumes were cooked from 20 min at atmospheric pressure.

The cooked material was frozen, freeze-dried and ground into flour (60 mesh). Resistant

starch values (Table 7) show that legumes presented lower RS values after thermal treatment

(4).

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Table 7 Resistant starch (RS) values of raw and freeze-dried cooked legumes (g/100g)

Legume RS (g/100 g)

Raw Cooked

Pea 2.45±0.30b 1.89±0.71

a

Common bean 3.72±0.79a 2.33±1.23

a

Chickpea 3.39±0.96ab

2.23±1.15a

Lentil 3.25±0.42ab

2.46±0.16a

Different letters in the same column indicate a statistical difference (p<0.05).

Data represent means and standard deviation (n=6).

2. Cooling

Cooling rate has an effect on resistant starch content in waxy maize starch. Waxy

maize starch dispersion were gelatinized and debranched by pullulanase (20 U/g) for 6 h.

After enzyme inactivation by heating, debranched starch was stored for 2 days at 4 or 20°C.

Debranched starch stored at 20°C had 50.1% RS, higher than that of starch stored at 4°C

which had 24.4% RS (9).

3. Heating-cooling cycle

Amylomaize VII starch dispersion (starch:water = 1:3.5) was autoclaved at 134°C for

1h, then cool and store at 4°C overnight. The autoclaving-cooling cycle was done for 0 to 4

cycles. Treated sample was then freeze-dried. Resistant starch content and thermal

characteristics of amylomaize VII were shown in Table 8. Increasing of autoclaving-cooling

cycle increased RS and transition enthalpy of the starch (19).

Table 8 Effects of autoclaving-cooling cycles on resistant starch yield and thermal

characteristics of amylomaize VII starch.

Number of

autoclaving-cooling

cycles

Resistant starch

yields (%)

Transition

temperature (Tp, °C)

Transition enthalpy

(∆H, J/g)

0 15.8 nd nd

1 21.3 149.3 2.7

2 25.2 149.6 4.5

3 29.9 148.1 7.0

4 31.8 152.9 8.8

Page 10: Resistant Starch

10

(A)

(B) (C)

(D) (E)

Fig. 5 Scanning electron micrographs of raw amylomaize VII starch (A), freeze-dried

amylomaize VII starch after one (B) and four autoclaving-cooling cycles (C),

oven- (D) and vacuum-dried resistant starch isolated from amylomaize starch after four

autoclaving-cooling cycles (E).

Scanning electron micrographs of raw, autoclaved-cooled starch and isolated RS were

shown Fig. 5. Raw amylomaize VII starch had a 5 µm starch granule. After autoclaving and

cooling, starch granule disappeared. Starch treated with 1 autoclaving-cooling cycle had a

irregular shaped particles with spongy-like porous network. A 4-cycle treated starch shown

more compact structure which related to higher melting enthalpy and its stabilization. For

isolated RS, porous structure was no longer visible. Oven-dried RS shown very compact and

dense particle. While vacuum dried RS formed an open, fluffy structure. Melting enthalpy of

vacuum-dried RS (28.7 J/g) was higher than oven-dried RS (19.7 J/g) because better

hydration capacity vacuum-drying (19).

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11

4. Debranching by pullulanase

Pullulanase or pullulan 6-glucanohydrolase (EC 3.2.1.41) is a debranching enzyme in

starch processing. It cleaves α-1, 6 linkages in pullulan, amylopectin and other related

polysaccharides, results in linear starch chains. Thus, provide an increased opportunity for

molecule alignment or aggregation to form crystalline structures, and hence, RS formation

(23).

Pongjanta et al. (2009) tested the effects of pullulanase concentration on RS content in

high amylose rice starch. High amylose rice starch dispersion (15% w/w) were annealed at

30°C for 1 h with vigorously shaking, then autoclaved at 121°C for 30 min and cooled to

55°C. The cooked starch samples were debranched using pullulanase enzymeat 0, 8, 10, 12,

14 and 16 U/g starch at 55°C for 16 h in a shaker water bath. The debranched samples are

then heated at 100°C for 15 min and stored at 4°C for 16 h. Afterward, a one cycle of

freezing (-10°C) and thawing (30°C) process of the samples was applied to promote syneresis

of the retrograded starches. The retrograded starch was dried at 45°C to approximately 13%

moisture content and grounded through 100-mesh sieve.

The retrogradation from the 8 to 12 U/g starch hydrolysis of the high amylose rice

starch was dramatically lost expressible water and remained relatively constant thereafter

with 12–16 U/g starch (Fig. 6A). Resistant starch content increased sharply as the amount of

the enzyme increased from 0 to 12 U/g starch because debranching increase opportunity for

crystallization of amylose molecules. However, increasing enzyme concentration from 12 to

16 U/g starch decreased RS content (Fig. 6B). Too long debranching process cause release of

small amylose molecules. Short amylose chains with DP 6–9 glucose units inhibit

retrogradation. While a chain length of at least 10 glucose units is required for crystallization

and formation of double helices, DP 20–30 is suitable to form RSIII. The results were in

accordance with hydrolysis rate of starches (Fig. 7). Among debranched starches produced in

this experiment, starch debranched by 12 U/g starch had the lowest hydrolysis rate, while that

of control was the highest. These results indicate that the incompletely-debranched RSIII

sample was resistant to α-amylase digestion (14).

Fig. 6 Effect of pullulanase enzyme concentration on degree of syneresis (A)

and resistant starch content (B) in RS III samples.

Page 12: Resistant Starch

12

Fig. 7 Effect of pullulanase enzyme concentration on α-amylase hydrolysis rate of

resistant starch type III samples, native high amylose rice starch (HARS),

commercial resistant starch (CRS; Hi-maize) and white bread (WB).

Resistant starch content was also affected by debranching time. In the study of Zhao

and Lin (2009) , 20% (w/v) maize starch dispersion was autoclaved at 121°C for 20 min and

cooled to 60°C. Then 1 ml of pullulanase solution (enzyme activity 30 PUN/ml) was added

and maize starch was kept at 60ºC in a water bath for 2 to 12 h with continuous agitation. The

starch paste was heated to 100ºC to inactivate the enzyme, then cooled to room temperature,

stored at 4ºC for 24h, before retreated with two autoclaving–cooling cycles. RS formation

was improved significantly as debranching time increased. The highest RS yield was

obtained with hydrolysis time of 12 h (Fig. 8a).

Fig. 8 Effects of pullulanase hydrolysis of gelatinized maize starch on RS formation.

a b

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13

However, when maize starch was treated with one autoclaving–cooling cycle, then

hydrolyzed by pullulanase at 60°C for different time durations, and followed by two

autoclaving–cooling cycles, the highest RS yield was obtained when retrograded maize starch

was hydrolyzed by pullulanase for 10 h. Prolonging hydrolysis of pullulanase could lead to

the decrease in RS yield as shown in Fig. 8b (23).

Similar results were also found in high amylose corn starch – Hylon V (about 55%

amylose) and Hylon VII (about 70% amylose). Amount of RS3 increased with debranching

time (Table 9). Hylon VII, which had higher amount of amylose, had higher amount of RS3

than that of Hylon V. However, peak transition temperature (Tp) and transition enthalpy (∆H)

of debranched starch were lower than that of native starch. Among debranched starch, peak

temperature was not different, but transition enthalpy increased with increasing debranching

time (12).

Table 9 Resistant starch (RS) contents and thermal properties of native and debranched

starch samples

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Health benefits of resistant starch

1. Resistant starch as a component of dietary fiber

By a definition of The American Association of Cereal Chemists (AACC), dietary

fiber is the edible parts of plants or analogous carbohydrates that are resistant to digestion and

absorption in human small intestine with complete or partial fermentation in large intestine.

When measure total dietary fiber by AOAC method, resistant starch is included in the result.

RS assays as an insoluble fiber but has the physiological benefits of soluble fiber. Like

soluble fiber, it has a positive impact on colonic health by increasing crypt cell production

rate or decreasing the colonic epithelial atrophy. It can also be used as vehicle for slow

release of glucose and reduction of serum cholesterol. Overall, it behaves physiologically as a

fiber (18).

2. Colonic production of short chain fatty acid (SCFA)

Resistant starch is the fraction of starch not hydrolyzed to D-glucose in the small

intestine within 2 hours, but is fermented in colon. RS is digested by bacterial amylases (i.e.

α-amylases, glucoamylase, isomaltase) and then glucose is metabolized into short chain fatty

acids (SCFA) such as acetate, butyrate, propionate and gases like CO2, H2, and CH4 etc. via

formation of pyruvate. 30 to 70% of RS is degraded to SCFA in the colon by bacterial

amylases.

SCFA produces an acidic environment which promotes the healthy bacterial

proliferation and inhibit pathogenic bacteria. It is a fuel for colonocytes (cells lining the

colon). It can increase colonic blood flow and electrolyte uptake, prevent development of

abnormal colonic cells. Among SCFA, butyrate appears to be the preferred substrate for

colonocytes and RS contributes high level of butyrate as shown in Table 10.

Table 10 Pattern of short chain fatty acids (SCFA) production from various substrates

Substrates Percentage of SCFA

Acetate Propionate Butyrate

Resistant starch 41 21 38

Starch 50 22 29

Oat bran 57 21 23

Wheat bran 57 15 19

Cellulose 61 20 19

Guar gum 59 26 11

Ispaghula 56 26 10

Pectin 75 14 9

From: Sharma et al. (2008)

3. Resistant starch and colorectal cancer risk

Cassidy et al. (1994) conducted a nutritional epidemiology ecological study which

gathered information on starch, NSP, fat and protein intake and mean national cancer

incidence rate in 12 countries (Table 11). Strong inverse associations between starch

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15

consumption and colon cancer (r = -0.76) and large bowel cancer (r = -0.70) incidence were

found (Fig. 9 and Table 11). While non-starch polysaccharide (NSP) also had inverse relation

but not significant. Fat and protein consumption increases risk of colorectal cancer. After

adjusting the effects of fat and protein intake, relationship still significant for RS and cancer

incidence (Table 12). The result suggested a potential protection role of resistant starch

against colorectal cancer and correspond with the hypothesis that fermentation in the colon is

the mechanism for prevention of colorectal cancer.(3)

Table 11 Dietary intake (g day-1) and cancer incidence (cases per 100,000 year-1

; age

standardized, world) in various populations

Fig. 9 The association between starch intake (g day-1) and colon cancer incidence (males and

females combined, n = 22) (cases per 100,000 age-standardised world population year-1).

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Table 12 Pearson correlation coefficients between dietary intake of starch, NSPs, protein and

fat and incidence of colorectal cancer .

Table 12 t-values of multiple regression analysis after adjusting for fat and protein intakes

and after interaction term analysis.

Leu et al. (2005) studied the synbiotic effect of RS and probiotics on colorectal cancer

risk. The 96 Male Sprague-Dawley rats with carcinogen-damaged cells in the colon were fed

with low or moderate RS diet (Table 13) and with or without Lactobacillus acidophilus or

Bifidobacterium lactis for 4 weeks.

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Table 13 Composition of experimental diet

The rats consuming the moderate-RS diet had higher (P< 0.001) total SCFA, acetate,

propionate, and butyrate concentrations in the feces compared with those fed the low-RS diet.

Probiotic bacteria supplementation did not affect fecal SCFA concentrations in rats fed either

the low or moderate-RS diets (Table 14).

Table 14 Effect of low- and moderate-RS diets supplemented with L. acidophilus (LA), B.

lactis (BL), or both (LA+BL) on fecal pH and fecal SCFA concentrations in rats.

Cecal pH was lower in rats fed the moderate-RS diets (P<0.001) compared with rats

fed the low-RS diets. Supplementation of probiotics to the RS diet did not affect pH. SCFA

concentrations in the cecum were greater in those fed moderate-RS diets (P<0.001). Probiotic

supplementation did not affect cecal SCFA concentrations (Table 15).

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Table 15 Effect of low- and moderate-RS diets supplemented with L. acidophilus (LA), B.

lactis (BL), or both (LA+BL) on cecal pH and cecal SCFA concentrations in rats.

The level of dietary RS affected total anaerobes (P=0.021), total aerobes (P<0.001),

lactobacilli (P<0.001), bifidobacteria (P<0.001), and total coliforms (P<0.001) in the cecal

digesta. Furthermore, the type of bacteria supplemented to the diet had a significant effect on

lactobacillus species numbers (P=0.004). B.lactis supplementation increased Lactobacilli

numbers (Table 16).

Table 16 Effect of low- and moderate-RS diets supplemented with L. acidophilus (LA), B.

lactis (BL), or both (LA+BL) on cecal microbial populations.

There was a significant interaction between the level of dietary RS and bacteria on the

acute apoptotic response to genotoxic carcinogen (AARGC) (P=0.002). The AARGC was

significantly elevated in the distal colon by B.lactis in rats fed RS, with or without L.

acidophilus (Fig. 10). L. acidophilus did not affect the AARGC nor did B. lactis in rats fed

low-RS. The level of dietary RS did not affect the AARGC (8).

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Fig. 10 Apoptotic index in the distal colon of rats fed a low-RS or moderate-RS diet

supplemented with L.acidophilus, B.lactis, or both.

4. Resistant starch and postprandial glycemia and hormonal response.

A study using 10 healthy normal weight males fed with test meals containing either

50g starch free of RS (0%RS), or 50g starch containing a high level of RS (54% RS) proved

the ability of high RS meals to significantly lower the postprandial concentration of blood

glucose and insulin (Fig. 11) (16).

Fig. 11 Change in plasma concentrations of glucose and insulin after a raw potato starch meal

(54% RS) (R) and a pre-gelatinized potato starch meal (0% RS) (S).

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5. Fecal bulking

A study on 11 healthy human consumed low- and high-RS diet (5 and 39 g RS/d,

respectively) found that high-RS diet caused a significant increase in fecal wet and dry

weight, and lowered fecal pH (Table 17). A significant correlation between RS consumed and

total output of feces (Fig. 12). For every 1 g RS consumed, fecal weight increase about 1.8 g

(13).

Table 17 Effect of a diet high in resistant starch (RS) on fecal output, pH, and starch and

nonstarch polysaccharide (NSP) concentrations1

Fecal variable Low-RS diet High-RS diet

Fecal output

(g wet wt/d) 138 ± 22 197 ± 372

(g dry wt/d) 38 ± 2 54 ± 72

Fecal pH 6.9 ± 0.1 6.3 ± 0.12

Fecal starch

(mg/g feces, wet) 10.0 ± 2.0 37.0 ± 7.02

(g/d) 1.7 ± 0.7 8.5 ± 2.02

Fecal NSP

(mg/g feces, wet) 65.5 ± 29.0 68.0 ± 27.0

(g/d) 7.6 ± 0.5 11.4 ± 0.12

1Mean ± SEM, n=11

2Significantly different from the low-RS dietary period, P < 0.01 (paired-difference t test)

Fig. 12 During the high-resistant starch (RS) dietary period there was a significant correlation

between dietary RS intake and fecal output (r = 0.70, P < 0.05).

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6. Resistant starch and lipid metabolism

Resistant starch has been found to affect the metabolism of low-density lipoproteins

(LDL), cholesterol, triglycerides, triglyceride-rich lipoproteins and total lipids. Soluble fibers

may bind bile acids or cholesterol in the intestine, preventing their reabsorption in the body.

The liver responds by taking up more LDL cholesterol from the blood stream thereby

lowering the concentration of LDL cholesterol in blood (18). A study in male rat fed with

cholesterol-free diet containing 50 g cellulose power (CP) or RS fraction from adzuki (AS) or

tebou (TS) starch found that RS diet caused lower in serum total cholesterol, HDL, IDL, LDL

and VLDL cholesterol (Fig. 13). Fecal total bile acid concentration in the AS and TS groups

were higher than the CP group (Table 18). The result suggested that serum cholesterol-

lowering effect of RS is due to the enhanced levels of hepatic SR-B1 and cholesterol 7α-

hydroxylase mRNA (7).

Fig. 13 Serum total cholesterol, VLDL + intermediate density lipoprotein (IDL) + LDL

cholesterol, and HDL cholesterol concentrations in rats fed with cellulose powder (�),

enzyme-resistant fraction of adzuki starch (�) or of tebou starch (◆) for 4 weeks.

Table 18 Fecal steroid concentrations in rats fed with CP, AS, or TS for 4 weeks.

CP, cellulose powder; AS, enzyme-resistant fraction of adzuki starch; TS, enzyme-resistant fraction of

tebou starch

Means within the same rows bearing different superscript roman letters are significantly different (P <

0.05)

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In conclusion, RS content in food varies by type and variety of raw food, and

processing conditions such as heating, cooling and debranching. RS has many health benefits

included reduction in risk of colon cancer, fecal bulking, modulation of glucose and lipid

metabolism and acting as a prebiotic.

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