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Page 1 of 23 Food & Function
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DOI: 10.1039/C6FO00661B
1 Physicochemical properties and digestibility of eleven Vietnamese rice starches with varying
2 amylose contents
3 Tien Dat Huynh1, Ashok K Shrestha2 and Jayashree Arcot1*

1
4 Food Science and Technology, School of Chemical Engineering, The University of New South
Wales, Sydney, NSW 2052, Australia,; 2School of Science and Health, Nutrition and Food
Published on 18 July 2016. Downloaded by Cornell University Library on 23/07/2016 21:35:01.
Food & Function Accepted Manuscript

6 Science, University of Western Sydney, Sydney, NSW 2751, Australia.
8 Authors’ email address: Tien Dat Huynh: tien.d.huynh@unsw.edu.au
9 Dr. Ashok Shrestha: A.Shrestha@uws.edu.au
10 Associate Professor Jayashree Arcot: j.arcot@unsw.edu.au
11 *Corresponding author: A/Prof. Jayashree Arcot, School of Chemical Engineering, The
12 University of New South Wales, Sydney, NSW 2052, Australia,. Ph: 61-2-9385 5360, Fax: 61-2-
13 9385 5966, Email: j.arcot@unsw.edu.au
14
1
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15 Abstract
16 The physicochemical properties of eleven Vietnamese rice starches with apparent amylose
17 contents ranging from 0.2% to 28.4% were investigated to identify the interplaying factors
18 contributing to the in vitro digestibility of starch: in uncooked, cooked in excess water and in
Hàm lượng AMY trong tinh bột gạo chi phối
19 limited water conditions. Amylose content of rice starches governed the physicochemical

đặc tính hóa lý cũng như độ trương nở, độ nhão và độ hồ hóa của tinh bột
20 properties of starch such as swelling power, pasting and gelatinization of starch which strongly
tương quan
21 correlated with raw starch digestibility. Amylopectin predominantly contributed to crystallinity
22 of starches both in short range and long range orders (observed by FT-IR and X-ray diffraction
23 respectively). This study found that cooking destructively changed the molecular structure of
24 starch that lead to increase in rate of starch digestion. Cooking in excess water resulted in a
25 higher rate of starch digestion at least by 2 fold in all examined starches, suggesting that in this
26 cooking condition factors that would normally limit the rate of digestion were eliminated.
27 Cooking in limited water that was similar to domestic cooking conditions, seemed to allow rice
28 starch granules to rearrange and interact together and limited the rate of digestion compared to
29 cooking in excess water. It can be concluded that physicochemical properties of starch can
30 predict the digestibility of raw starches only but not cooked starches. The digestion of rice starch
31 cooked in limited water cannot be predicted by just studying the uncooked starches or cooking in
32 excess water.
33
34 Keywords
35 Starch, physicochemical, digestibility, cooking
36
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37 1 Introduction
38 Rice (Oryza sativa, L.) is one of the main staple foods for a majority of the Asian population and
39 is eaten as whole cooked grain, mostly by steaming or sometimes followed by stir-frying.
40 Studies have shown that the degree of starch digestion varies between starchy foods, suggesting
41 the choice of foods with lower glucose response so as to prevent type 2 diabetes, obesity and

42 metabolic syndrome 1. Under this vision, the rate at which the glucose is released from rice
43 starch digestion has been extensively investigated. It is generally assumed that physicochemical
44 properties of starch such as amylose content, gelatinisation and pasting properties, degree of
45 crystallisation, swelling power and granular surface short range order structure, amylopectin fine
46 structure, etc. strongly correlated with rice starch digestion in form of cooked 2-6 or uncooked
47 rice 7, 8. Implications based on relationships between physicochemical properties and digestion of
48 rice starch seem to be oversimplified and maintained unclear, may somehow mislead customer’s
49 choice of rice varieties having low starch digestibility. In addition, majority of studies were very
50 limited in which only few varieties have been studied with different amylose contents, and thus
51 drawn conclusions seem to be generalised for other varieties. The extent of starch digestibility
52 has been reported to be dependent on cooking methods 9-11. Most of the studies have addressed
53 rice starch digestibility of rice cooked in excessive water, however, practically, rice is mostly
54 cooked in limited water, especially in a rice cooker. Also, the existing literature has raised
55 doubts about whether uncooked starch digestion can predict cooked starch digestion, particularly
56 in limited water conditions. In this paper, a comprehensive study on physicochemical properties
57 of eleven rice starches isolated from eleven rice varieties with a wide range of amylose contents
58 and their starch digestibilities in the forms of uncooked, cooked in excess water and in limited
59 water was performed to clarify their relationship.
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60 2 Materials and methods
61 2.1 Materials
62 Enzymes and chemicals were obtained from the following sources: pepsin (Sigma P-6887, from
63 gastric porcine mucosa), pancreatin (Sigma P-1625 from porcine pancreas) and
64 amyloglucosidase (Novozyme, 300L AMG). Rice varieties e.g., Jasmine, VD20, OM6976,

65 IR50404 and OM4218 were supplied by An Giang Plant Protection Company (An Giang,
66 Vietnam), other rice varieties (provide details here) were collected from Nam Dinh province,
67 Vietnam.
68 2.2 Starch isolation
69 Rice starch was isolated from rice grains using the alkaline steeping according to the method of
70 Wang and Wang12 with some modifications. Rice grains (100g) were soaked in 200 ml of 0.1%
71 NaOH for 18 hours, blended using Thermomix (Thermomix, Australia) for 2 minutes at high
72 speed. The starch slurry was passed through a micro-sieve 63µm and centrifuged at 1400g for 10
73 min. The supernatant was removed. The starch layer was washed with deionized water and
74 centrifuged. Then the starch was re-slurried and neutralized with 1.0M HCl to pH 6.5 and
75 centrifuged. Starch was washed with deionised water at least 3 times before drying at 45oC for
76 48h, passed through 200µm sieve and stored at room temperature. The isolated starches were
77 tested for apparent amylose content following the AACC 61-03 method13. The isolated starches
78 were tested for physicochemical properties and in vitro digestion.
79 2.3 Swelling power (SP) and pasting properties of rice starches (RVA)
80 Swelling power of starch was determined based on the method developed by Tsai, Li and Lii 14.
81 The SP was calculated using the formula: SP = Ws/ [0.1(100% - WSI)] (g/g), where WSI is the
82 water soluble index (WSI = [W1/0.1] x 100%), W1 is the soluble starch fraction and Ws is the
83 sediment.
84 Pasting properties of rice starches were determined by using the Rapid Visco Analyser (RVA
85 Series 1, Newport Scientific, Warriewood, Australia) following the AACC 61-0213. The RVA
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86 graphs were generated and the results were calculated by Thermoline software 2.2 (Newport
87 Scientific, Warriewood, Australia).
88 2.4 Rice starch thermal properties (DSC)
89 Differential scanning calorimetry (DSC) Q20 Series (TA Instrument, New Castle, DE) was used
90 to investigate thermal properties of rice starches. Around 3 mg of sample was mixed with water

91 in the ratio 1:2.3 and hermetically sealed in an aluminum pan. The pan was equilibrated at room
92 temperature at least 2 hours before running the DSC. Samples were heated from 30 to 100oC at
93 the rate of 10oC/min. The temperature and transitions were recorded and calculated by TA
94 Universal Analysis Software version 4.5.
95
96 2.5 X-ray diffraction (XRD) and relative crystallinity of rice starches
97 PANalytical Xpert Multipurpose X-ray Diffraction System (MP XRD, X′ Pert Pro, PANalytical,
98 Netherlands) was used to observe the crystalline pattern of starch samples. Starches (12%
99 moisture content) were measured in triplicate with target voltage around 40kV, target current of
100 45 mA. The instrument was equipped with a copper X-ray generator (λ = 1.54 Å), programmable
101 incident beam divergence slit and diffracted beam scatter slit, and an X’celerator high speed
102 detector. X-ray diffractograms were acquired at room temperature over the 2θ range of 6–50°
103 with a step size 2θ of 2.0°/min. The relative crystallinity of the starches was quantitatively
104 calculated using peak-fitting technique15 by using MagicPlot 4.0 software (MagicPlot, MagicPlot
105 Systems, LLC, St. Petersburg, Russia).
106 2.6 Fourier Transform Infrared (FTIR) spectroscopy
107 FT-IR of rice starches (12% moisture content) were performed by using a Bruker IFS66/S
108 Fourier Transform Infrared spectrometer equipped with a tungsten halogen lamp, a KBr beam
109 splitter, ATR single crystal and a DTGS detector. The spectra in the region of 1200-800 cm-1
110 were recorded from the co-added interferograms of 32 scans with resolution of 4 cm-1. The IR
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111 absorbance values at 1047 and 1022 cm-1 were extracted from the deconvoluted spectra and their
112 ratios were calculated.
113 2.7 In vitro digestibility of rice starch
114 The protocol of in vitro digestion described by Muir and O’Dea16 was used with some
115 modifications. For uncooked rice starches, 100mg of starch (in dried weight) was transferred into

116 120mL plastic container (Sarstedt, Australia), 1 magnetic bar and 10mL of HCl 0.02M were
117 added. The gastric phase was mimicked by adding 5mL of pepsin solution in HCl 0.02M
118 (1mg/mL). The containers were placed in a sample holder of the Glycemic Analyzer (GI20, Next
119 Instrument, Australia) for 30 min operating at 37oC with stirring speed of 200 rpm. After 30 min,
120 the solution was neutralized by 15mL of 0.02M NaOH and 25mL of acetate buffer (0.2M, pH 6)
121 was added to each container. The containers were equilibrated at 37oC for 5 min before adding
122 5mL of enzyme mixture (2mg/mL of pancreatin; 28U/mL amyloglucosidase). The containers
123 were incubated for 5 hours and the hydrolysates were taken with different time intervals.
124 Glucose released was measured using the glucose analyser (GL6, Analox, India). For the cooked
125 rice starch in excess water, the procedure for cooking was according to Reed, Ai, Leutcher and
126 Jane10 with modifications. 100mg of rice starch (dried weight) was transferred into a 120mL
127 plastic container and cooked with 5mL of deionized water in boiling water bath (Ratek
128 8WB20D, Edwards Group Pty Ltd. Narellan NSW 2567, Australia) for 10 min. The temperature
129 of cooked starch was brought to 37oC and immediately adjusted to a total volume of 10mL with
130 0.02M HCl and the digestion was done following the aforementioned procedure. Rice starches
131 were also cooked by simulating the conditions of cooking for normal consumption which was
132 modified from 17 procedure. 3 grams of rice starch was weighed into a 15mL glass container with
133 screw cap and water was added in the ratio of 2:3 and 1:1 for non-waxy and waxy rice starches,
134 respectively. All glass containers were placed in a rice cooker (Kambrook Electric, NSW,
135 Australia) containing 200 grams of white rice and 300 grams of water. Rice starch was cooked in
136 an electric rice cooker with a fixed pre-determined time. 100mg of freshly cooked starch (dried
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137 weight) were transferred into a 120mL plastic container and immediately digested using the
138 same in vitro digestion procedure described above.
139 2.8 Microscopy
140 The dried uncooked starches and residues after digestion were spread on 26 mm diameter
141 circular metal stubs coated with dual-sided adhesive carbon. The stubs were transferred into a

142 humidity oven and kept overnight at room temperature. The samples were gold coated by
143 Emitech K550X Sputter Coating Systems (Emitech, England) under vacuum and argon
144 environment (5 min at 125mA), yielding around 50 nm coating thickness. Coated samples were
145 imaged in a scanning electron microscope (SEM), Hitachi S-3400 (Tokyo, Japan) at an
146 accelerating voltage of 15-20 kV and a working distance of 10 mm.
147
148 2.9 Data fitting and statistical analysis
149 The first-order model (Eq.(1)) developed by Goñi, Garcia-Alonso and Saura-Calixto18 was used
150 to investigate the kinetics of rice starch digestion that mimics the small intestinal conditions.
151 C = C∞ (1- e-Kt) (1)
152 Where C = starch digested (expressed as % db) at incubation time t (min), C∞ = equilibrium
153 starch digested (express as % db), K = digestion rate coefficient (min-1). K values were obtained
154 by a nonlinear-least-squares fit of the Eq. (1) using GraphPad Prism 6.0 (GraphPad Software,
155 Inc.)
156 The data are reported as means with standard deviations of triplicate measurements. Analysis of
157 variance (ANOVA) with Duncan’s multiple tests and Pearson correlation were determined by
158 SPSS version 22 (SPSS Inc.)
159
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160 3 Results and discussion
161 3.1 Apparent amylose content
162 Amylose is an important component of the rice starch granule, affecting cooking and sensory
163 qualities, physicochemical properties and bioavailability of starch. Apparent amylose content of
164 Vietnamese rice starches in this study ranged from 0.2 to 28.4 % (Table 1). According to

165 Mackill, Coffman, and Garrity 19, based on amylose content, these varieties can be classified
166 into: waxy (0-2% amylose in white glutinous and Cai Hoa Vang), low amylose (8-20% in Bac
167 Thom, Jasmine and VD20), intermediate amylose (21-25% including Tam Xoan, OM4218) and
168 high amylose (>25% including OM6976, IR50404, Luong Quang and Q5). The range of amylose
169 content in this study is quite different from that reported earlier20 (1.9-39.7%). Variation in
170 reported amylose values is most likely due to the difference in analytical method used. Various
171 factors affecting the amylose content have been extensively reviewed21. For example, amylose
172 contents of Cai Hoa Vang, Tam Xoan and Q5 were reported as 2.8, 26.8 and 36.2% respectively
173 in another study20 using the Rice analyzer, which were higher than that measured in the present
174 study.
175 3.2 Swelling power of starch (SP)

khả năng trương nở
176 Swelling power of Vietnamese rice starch was calculated and is presented in Table 1. The SP of
bởi 2 xu hướng khác nhau cho tinh bột gạo nếp
177 Vietnamese rice starches were characterized by two distinct trends. For waxy rice starches, the
khả năng trương nỏ tăng nhanh chóng ở 55-75 độ và sau đó đạt ổn định tại 95 độ
o o
178 SP values were rapidly increased between 55 C and 75 C and followed by a steady growth in SP,
mặt khác khả năng trương nỏ của bột gạo tẻ tăng chậm ở nhiệt độ 55-85 độ
o
179 reaching over 30 (g/g) at 95 C. On the other hand, SP of non-waxy rice starches slowly increased
nhưng tăng rõ rệt sau khi đạt 85 độ. Những sự thay đổi nhỏ về khả năng trương nở ở
180 between 55 and 85oC but remarkably increased after reaching 85oC. Minor changes in SP
nhiệt độ từ 65-75 độ được tìm thấy ở cả hai tinh bột gạo tẻ và gạo nếp hàm lượng AMY
181 between 65 and 75oC were found in both waxy and non-waxy rice starches. The amylose content
của gạo liên quan đáng kể đến khả năng trương nỏ của tinh bột gạo kết quả này đã xác nhận hành vi trương nỏ của tinh bột gạo
182 of rice was significantly associated with SP of rice starches. These results confirmed the swelling
như đã báo cáo ở trước nhìn chung tinh bột gạo nếp có khả năng trương nỏ cao hơn tinh bột gạo tẻ
183 behavior of rice starches as previously reported7, 22. Generally, waxy rice starch had higher SP
tại giai đoạn nấu đầu tiên tuy nhiên, khi tinh bột gạo tẻ được nấu tại nhiệt độ cao 95 dộ
184 than non-waxy rice starch at the first stage of cooking. However, when non-waxy rice starch was
khả năng trương nỏ của gạo tẻ tiệm cận với gạo nếp chỉ ra rẳng
185 cooked at a high temperature (95oC), the SP values approached those of waxy rice, indicating
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cấu trúc của cả tinh bột gạo nếp vè gạo tẻ đều có thể bị phá vỡ
ở mức độ tương tụ nhau khi
186 that both waxy and non-waxy rice starches’ structures may be disrupted to a similar extent when
được nấu chín trong nước sôi dư thừa Amylopectin góp phần đáng kể vào hành vi trương nở của tinh bột ngũ cốc,
187 being cooked in excess boiling water. Amylopectin significantly contributed to swelling
trong khi amyloza đóng vai trò như một chất ức chế trương nở.
188 behavior of cereal starches, whereas amylose acted as a swelling inhibitor 23. It is possible that
Có thể việc nấu trong nước thừa ở nhiệt độ cao có thể làm mất vai trò ức chế của amyloza,
189 cooking in excess water at high temperatures may negate the inhibitory role of amylose, making
làm cho các loại tinh bột không sáp nở tự do hơn
190 non-waxy starches swell more freely.
9
Published on 18 July 2016. Downloaded by Cornell University Library on 23/07/2016 21:35:
191 Table 1 Swelling power and RVA parameters of rice starches A,B
Swelling powers RVA parameters

Starch
Amylose Peak Breakdown Final Setback Pasting
samples 55oC 65oC 75oC 85oC 95oC
(RVU) (RVU) (RVU) (RVU) temp. (oC)

Q5 tẻ 28.4 ± 0.5i 3.6 ± 0.2cd 9.6 ± 0.1c 13.5 ± 0.2bc 13.7 ± 0.2ab 20.6 ± 1.3a 257.5 ± 2.2f 38.5 ± 1.7a 332.2 ± 2.8h 113.1 ± 6.7e 90.3 ± 0.4g
Luong Quang tẻ 27.5 ± 0.3h 3.7 ± 0.2d 8.8 ± 0.1c 12.3 ± 0.5a 13.3 ± 0.7a 30.7 ± 0.9de 280.6 ± 2.9h 45.8 ± 0.2b 349.5 ± 5.4j 114.8 ± 2.2e 91.3 ± 0.4h
IR50404 tẻ 26.9 ± 0.1g 3.3 ± 0.2ab 3.4 ± 0.0a 14.8 ± 2.0cd 16.0 ± 0.4c 26.4 ± 1.9b 270.2 ± 1.7g 49.6 ± 2.3b 340.5 ± 2.4i 120.9 ± 4.5f 82.2 ± 0.3e
OM6976 25.1 ± 0.1f 3.3 ± 0.1ab 3.8 ± 0.0a 13.2 ± 1.4bc 16.7 ± 0.6cd 31.0 ± 0.9de 225.2 ± 1.3c 89.9 ± 3.2de 270.5 ± 1.1e 132.5 ± 0.8g 87.6 ± 0.3f
OM4218 23.8 ± 0.4e 2.9 ± 0.3a 3.8 ± 0.2a 15.2 ± 0.4d 18.6 ± 0.5e 37.2 ± 0.8f 231.5 ± 0.4d 82.3 ± 2.4d 273.3 ± 3.7e 124.0 ± 0.9f 79.6 ± 0.5d
Tam Xoan tẻ 23.5 ± 0.3e 3.5 ± 0.3cd 3.9 ± 0.1a 12.9 ± 0.5ab 14.2 ± 0.2b 27.4 ± 1.0c 244.6 ± 0.8e 83.8 ± 1.5d 308.6 ± 2.2g 150.0 ± 1.5h 91.3 ± 0.4h
VD20 16.4 ± 0.3d 3.1 ± 0.1ab 9.4 ± 0.7c 15.0 ± 0.2cd 18.9 ± 0.3e 27.3 ± 1.9c 260.7 ± 0.6f 61.1 ± 5.9c 297.0 ± 3.4f 97.4 ± 1.9d 75.3 ± 0.1c
Jasmine 15.3 ± 0.2c 3.6 ± 0.6cd 8.9 ± 0.1c 13.7 ± 0.3cd 17.3 ± 0.5d 30.8 ± 1.1de 233.8 ± 1.7d 87.6 ± 0.2d 244.7 ± 2.2d 98.5 ± 0.3d 87.1 ± 0.1f
Bac Thom 10.4 ± 0.2b 3.3 ± 0.0abc 6.9 ± 0.2b 14.3 ± 0.1cd 18.9 ± 0.2e 30.0 ± 2.1d 296.0 ± 7.6i 116.8 ± 5.5f 273.6 ± 2.4c 58.4 ± 0.3c 75.4 ± 0.1c
Cai Hoa Vang nếp 0.5 ± 0.1a 4.8 ± 0.1f 20.0 ± 0.8e 29.5 ± 1.4e 30.5 ± 0.1f 33.4 ± 0.3e 201.0 ± 0.5a 113.5 ± 3.5f 107.0 ± 1.9a 21.2 ± 1.6a 68.1 ± 0.1a
White glutinous nếp0.2 ± 0.1a 4.2 ± 0.4e 17.6 ± 1.0d 28.9 ± 0.3e 31.8 ± 1.0f 38.2 ± 0.5f 214.6 ± 0.9b 119.5 ± 1.3f 123.1 ± 0.6b 28.0 ± 2.2b 69.9 ± 0.1b
192 A Values are means ± standard deviation of triplicates
B
193 Values with different superscripts in each column are significantly different at p<0.05 determined using SPSS, version 22
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194 3.3 X-ray diffraction
195 The supra-molecular structures of rice starches were characterized using the X-ray diffraction technique.
196 The selective diffractograms of rice starches and degree of crystallinity are displayed in Fig 1.
197 Vietnamese rice starches showed a typical A-type diffraction pattern with crystalline peaks at 15o, 17o,
198 18o, 20o and 23o (Fig. 1). The XRD patterns were quite similar between starches though differences in
199 peak intensities were recorded. Differences in intensity reflect the variation in degree of crystallinities

200 among tested starches. The degree of crystallinity ranged from 45.9% to 71.1%. The amylopectin
201 contents of starches were proportional to the degree of crystallinity. White glutinous rice had the highest
202 and Q5 starch had the lowest crystallinity. The crystalline structure of starch granules is formed by the
203 packing of double helices of amylopectin (long range ordered structure) 24. Higher amount of
204 amylopectin is expected to have higher crystallinity as observed. The relative crystallinities found in this
205 study is much higher than that previously reported values7. An alternative fitting method15 was used in
206 which crystalline peaks were deconvoluted and the relative crystallinity was calculated based on each
207 individual peak area, resulting in higher and more accurate prediction of crystallinity of starch.
208
209 Fig. 1 X-ray diffractogram (crystallinity) of selected Vietnamese rice starches. Crystalline peaks in
210 degree and percentage relative crystallinity of individual rice starches are shown. Data have been
211 offset for clarity
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212 3.4 Fourier transform infrared (FTIR) spectroscopy
213
White Glutinous
0.8 A VD20 B
Tam xoan
Q5
Luong Quang
0.6
Relative absorbance
Jasm ine
Cai Hoa Vang
Bacthom
0.4 OM6976
OM4218
IR504

0.2
0.0
1200 1100 1000 900 800
Wavenumber (cm-1)
214
215 Fig.2 FT-IR spectra of raw rice starches (A) presented from 800 to 1200 cm-1and 1047/1022cm-1
216 ratios (B). The bar graph shows the trend of decreasing amylose content from left to right
217 The infrared spectroscopy was used to explore the short range molecular order structure near the granule
218 surface of rice starches. The FT-IR spectra of rice starches, from 800-1200cm-1 (Fig. 2A) appeared to be
219 similar. Van Soest, Tournois, de Wit, and Vliegenthart 25 reported that FT-IR bands at 1047 cm-1 and
220 1022cm-1 denote ordered and amorphous regions, respectively. The ratio of the intensity of these bands
221 (1047/1022) was used as a convenient index to exemplify the degree of short range order of the starches
222 (crystalline of double helices). 1047/1022 ratios of rice starches are presented in Fig.2B. Waxy rice
223 starches had the highest 1047/1022 ratios among tested rice starches that were in agreement with a
224 previous study 7. It is quite clear that amylopectin predominantly denotes amount of double-helix in
225 waxy rice starches. Q5 and Luong Quang starches (highest in amylose contents) were characterized with
226 very low 1047/1022 ratios (0.69 and 0.67, respectively). However, in high amylose starches (>20%),
227 FT-IR ratios were not necessarily low. For example, OM4128 was relatively high in 1047/1022 ratios
228 (0.72) although its amylose content was 23.8%, indicating the contribution of amylose in forming
229 double helices of this starch.
230 3.5 Pasting properties of rice starch

sự hồ hóa tinh bột gạo có mặt nhiệt độ, nước và lực cắt dẫn đến hiện tượng nhão
231 Gelatinization of starch granules in the presence of heat, water and shear resulted in pasting behaviors,
được đo bằng máy phân tích nhanh độ nhớt đặc tính hồ (past) của tinh bột gạo
232 as measured by the Rapid Visco Analyser (RVA). The pasting properties of Vietnamese rice starches are
tinh bột gạo được đặc trưng bởi 2 biên dạng RVA riêng biệt giữa
233 shown in Table 1. Vietnamese rice starches were characterized by two distinct RVA profiles between
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gạo nếp và gạo tẻ tinh bột gạo đỉnh nhớt, độ nhớt thiết lập lại và độ nhớt cuối cùng được tìm thấy ở
DOI: 10.1039/C6FO00661B
234 glutinous and non-glutinous rice starches. Low peak, setback and final viscosities were found in White
tinh bột gạo nếp (Cái Hoa Vàng và White) theo báo cáo này, các hạt tinh bột gạo nếp dễ rã và kém cứng hơn
235 and Cai Hoa Vang glutinous rice starches. It is reported that easily disintegrated and less rigid glutinous
có thể làm giảm khả năng chịu lực cắt kết quả giảm độ nhớt đỉnh
236 rice starch granules may reduce the resistance to shear force; consequently decreasing peak viscosity 22.
nhệt độ nhão của hồ (past) của tinh bột gạo VN trong khoảng từ 68-91 độ
237 The pasting temperature of Vietnamese rice starches ranged from 68-91oC (Table 1). Pasting
tinh bột gạo nếp Cái Hoa Vàng và cao nhất trong tinh bột gạo tẻ LQ và TX
nhiệt độ hồ (past) được tìm thấy là thấp nhất trong
238 temperature was found to be lowest in Cai Hoa Vang glutinous rice starch and highest in Luong Quang
phát hiện này xác nhận rằng tinh bột gạo nếp dễ bị hồ hóa hơn
239 and Tam Xoan starches. This finding confirms that glutinous rice starch is more prone to gelatinization
tinh bột gạo tẻ giá trị độ nhớt thiết lập lại trong nghiên cứu này là cao nhất đối với tinh bột có hàm lượng AMY cao
so với

240 compared to its non-glutinous counterpart. Setback values in the current study were highest in high
và thấp nhất đối với tinh bột gạo nép
241 amylose rice starches (Luong Quang, Q5, and IR50404) and lowest in glutinous rice starches (Cai Hoa
Có khảng năng là một lượng lớn AMY được tách ra từ các loại tinh bột có hàm lượng AMY cao
242 Vang and White Glutinous). It is likely that a high amount of amylose leached from high amylose
và tổ chức lại trong quá trình làm nguội cho nên mà độ nhớt thiết lập lại của các giống gạo này sẽ cao tuy nhiên, trong số tinh bột gạo tẻ
243 starches and reorganized during cooling, resulting in a high setback. However, among non-waxy rice
giá trị độ nhớt thiết lập lại cũng có thể ảnh hưởng bởi khả năng tách ra của AMY từ tinh bột gạo
244 starches, setback values may also be influenced by the leaching ability of amylose from the starch
bất kể hàm lượng AMY của chúng là bao nhiêu có thể là một lượng lớn AMY thoát ra
7
245 granules regardless of their amylose contents . It is possible that the high amount of amylose leaching
từ tinh bột có hàm lượng AMY trung bình cũng gây ra giá trị độ nhớt thiết lập lại co như được quan sát thấy trong tinh bột gạo Tam Xoan
246 from an intermediate amylose starch also causes exceptionally high setback as observed in Tam Xoan
247 rice starch (150 RVU) in this study.
248 3.6 Thermal properties of rice starches
249 Gelatinization properties of Vietnamese rice starches were determined by DSC. The DSC parameters of
250 endotherms associated with gelatinization of Vietnamese rice starches are presented in Table 2.
tinh bột gạo nếp có đỉnh thu nhiệt rộng rõ rệt
251 Glutinous rice starch (Cai Hoa Vang and White glutinous) had distinct broad endothermic peaks
trong khi đỉnh nóng chảy nông được phát hiện ở các guống có chứa hàm lượng AMY cao nhất
252 whereas shallow melting peaks were detected in varieties containing the highest amylose starches
Xem hình 3A cho các loại tinh bột khác xu hướng đỉnh thu nhiệt cao hơn và sắc nét hơn
253 (Luong Quang and Q5) (Fig.3A). For other starches, a trend of sharper and higher endothermic peak
được tìm thấy tương ứng với việc tăng hàm lượng AMY đặc biệt là TX, 504, OM4218
254 was found corresponding to increasing amylose content, especially in Tam Xoan, IR50404, OM4218
Xem hình 3B nhiệt độ chuyển tiếp
255 and OM6976 starches (Fig.3B). Temperatures of transition including onset (To), peak (Tp), conclusion
khoảng nhiệt độ hồ hóa
256 (Tc), gelatinization temperature range (∆T= Tc-To) and gelatinization enthalpy (∆H) are presented in the
phạm vi nhiệt độ chuyển tiếp
257 supplementary Table S1. DSC endothermic parameters varied between starches. The range of transition
258 temperatures were To, 52.1-66.9oC; Tp, 65.6-75.4oC; Tc, 78.1-85.9oC; ∆T, 19.3-33.5oC; and ∆H was 9.2-
giá trị ..... cao nhất được tìm tháy trong tinh bột gạo nếp
259 13.0 (J/g). The highest ∆H and ∆T values were found in waxy rice starches. Endothermic transition has
260 been reported as the loss of crystallites which were mainly formed by amylopectin 26. High degrees of
261 crystallinity found in Vietnamese waxy rice starches (Fig.1) explained their relatively high
To: nhiệt độ bắt đầu phản ứng hồ hóa
Tp: nhietj độ phản ứng cực đại
Tc: nhiệt độ kết thúc phản ứng
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262 gelatinization enthalpies. In addition, large gelatinization temperature range of these starches may have
263 also amplified gelatinization enthalpies since transition temperature range is proportional to enthalpy.
264 On the other hand, amylose is associated with increase in heat capacity of starch 27 and may somehow
265 increase gelatinization enthalpies. However, this trend was not observed in Luong Quang and Q5
266 starches (amylose >26%, Table 1) which had significantly lower gelatinization enthalpies. It is possible
267 low gelatinization enthalpies in these starches could be because of their low short range order

268 architecture. It is reported that the gelatinization enthalpy is proportionate to the amount of short range
269 order 28. The significantly (p<0.05) low 1047/1022 ratios found in Luong Quang and Q5 starches may
270 lower their gelatinization enthalpies.
A B
271
272 Fig. 3 Differential scanning calorimetry curves of (A): low Tp starches and (B): high Tp starches.
273 Data have been offset for clarity
274 3.7 In vitro digestion of rice starches
275 3.7.1 In vitro digestion of uncooked rice starches
276 In vitro digestion of starches in uncooked, cooked in excess water and limited water were conducted.
277 Digestion rate and extent for uncooked rice starches are presented in Table 2. Digestion behaviors of
278 starches precisely followed first-order and fit the non-linear equation Eq.1. (R2> 0.95, the data not show
279 here). The fitted digestograms are plotted and presented in Fig.S1A. Highest rates of starch digestion
280 were found in Cai Hoa Vang [(10.39 ± 0.29) x 10-3] and White Glutinous [(10.02 ± 0.53) x 10-3] starches
281 in which most of their starches were hydrolyzed within the first 180min. As visualized by SEM
282 imaging, Cai Hoa Vang starches (Fig.4A) were damaged by amyloytic enzyme after 1 hour and very
283 small fragmented residues were observed after 3 hours. In contrast, IR50404, OM6976, OM4218 and
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284 Tam Xoan starches were characterized with a very low rate and extent of digestion (Table 2) and
285 showed a high resistance to starch hydrolysis. These starches were gradually hydrolyzed by α-amylase
286 over the digestion time course (Fig. S1A). SEM imaging also demonstrated that OM4218 starch
287 granules were less degraded and no cavity was found on granular surface. In comparison, other starches
288 showed intermediate level of starch digestibility, that is, in Cai Hoa Vang and OM4218 starches.
289 Cavities were found on surface of these starches after 60 min digestion (Fig.4B, Fig.4D) as proof of rate

290 of enzymatic attack compared to other starches.
291 The relationship between the digestion rate coefficient of raw starch and physicochemical parameters
292 have been analyzed and presented in Table S2. The digestion rate coefficient negatively correlated with
293 amylose content of tested starches. However, starches containing the highest amylose content do not
294 necessarily have the lowest digestion rate coefficients. For example, Q5 and Luong Quang had the
295 highest amylose contents (28.4 and 27.5% respectively) but were characterized with intermediate rate
296 coefficients. In fact, the presence of supra-molecular arrangement of amylose in starch granules are most
297 likely governing the physicochemical properties of starches and would indirectly be associated with
298 amylolytic susceptibility. Firstly, amylose content contributed to forming compact and stable native
299 starch granules resisting amylase hydrolysis. The structure stability was clearly reflected in the
300 resistance to swelling and gelatinization (especially low in SP, high in pasting temperature, onset of
301 gelatinization and peak gelatinization temperatures) as seen in IR50404, OM6976, OM4218 and Tam
302 Xoan starches. Thus, the correlations between digestion rate coefficients of uncooked rice starches and
303 physicochemical parameters were established as indicated in Table S2. On the other hand, the amylase
304 susceptibility of native starches is reported to be affected by organization of the starch granular surface
29
305 . It is generally accepted that a higher FT-IR ratio of starch (1047/1022cm-1) should result in greater
306 resistance to starch digestion25, 29. However, this relationship was not observed in this study. According
307 to Jenkins and Donald30, the presence of amylose may form a co-crystalline network between amylose
308 and amylopectin. It is possible that only ordered structures derived from amylopectin and reinforced by
309 amylose can make the starch granular surface more stable and less porous; resulting in less starch
310 hydrolysis. For example, OM4218 had the highest 1044/1022 cm-1 ratio (0.72) among the non-waxy rice
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311 starches and was more resistant to amylase hydrolysis. The lack of surface cavities caused by enzyme
312 attack as found in Fig.4C strengthens this hypothesis.
313 3.7.2 In vitro digestion of cooked starch in excess water
314 In this experiment, the rice starches were cooked in excess water and their starch digestibilities were
315 investigated. Table 2 showed that cooking in excess water significantly increased the digestibility of all
316 rice starches. The non-linear first order fit of digested starch by time (Fig.S1B) and the extent of

317 digestion and rate coefficients is present in Table 2. The digestibilities of cooked rice starches were
318 much higher compared to that of raw starches in which the extent of digestion in the first 30min
319 increased 2-7-fold and the rate coefficient increased up to 8 fold. Only minor, although statistically
320 significant (p<0.05), differences were found in the rate and extent of starch digestion between samples
321 cooked in excess water (Table 2). The results indicate that the molecular arrangement of starch acted as
322 a rate limiting step and was possibly disrupted by cooking making starches more susceptible to
323 amylolysis. The relationship between physicochemical properties and rate coefficients were further
324 analyzed for cooked starches in excess water and shown in Table S2. Although very similar
325 relationships were found, the correlation and degree of significance decreased, in comparison to raw
326 starches. Our results indicate that physicochemical parameters more closely correlate to native starch
327 digestion rather than cooked starch. However, caution in interpreting correlations between
328 physicochemical parameters and cooked starch digestibility should be taken. For example, IR50404
329 starch was high in amylose (27%) but its starch digestibility was also very high when being cooked in
330 excess water.
331 3.7.3 In vitro digestion of cooked starches in limited water
332 Considering that rice is always eaten in cooked form mostly using the absorption method with limited
333 water to rice ratio. In the current study, rice starches were cooked in a way that mimicked domestic
334 cooking using an electrical rice cooker. The behavior of digestion was studied and is presented in Table
335 2 and Fig.S1C. Generally, starch digestibility was highly variable between samples and the rate of
336 digestion significantly decreased (p<0.01), compared to that of starch cooked in excess water. The
337 cooking conditions probably allowed starch granules to swell and interact with adjacent granules. When
338 heating high starch concentration solutions (20% w/w starch to water), the gel network may be formed
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339 (mainly by amylose) and reinforced by interaction between swollen starch granules 31. This sort of gel
340 did not exist when starch was cooked in excess water since the concentration of starch was too low. The
341 rearrangement of amylose and amylopectin at molecular and granular levels could significantly
342 influence the amylolytic susceptibility of the cooked rice starches. At the molecular level, both amylose
343 and amylopectin may form a gel structure between high molecular weight polymer molecules following
344 further increase in crystallinity in the polymer rich regions. The formation of such gels, in turn, act as a

345 barrier for dispersion of starch granules and restrict the rate of starch digestion 17. The digestibility of
346 cooked rice starch in limited water was found to be independent of physicochemical properties (Table
347 S.2). The retrogradation of the starch gel was strongly influenced by physicochemical properties of
348 starch32, 33 and was thought to have a good correlation with digestibility of starch. However, the starch in
349 this study was freshly cooked and digested, which minimized retrogradation as a consequence. Factors
350 other than amylose such as interactions between the leached components of starch and amylopectin fine
351 structure (not included here) influence the digestibility of cooked starch in limited water. The correlation
352 between digestion rate coefficient of starch in the raw, cooked in excess water and cooked in limited
353 water conditions were investigated (Fig.S2). Though, statistically significant (p<0.05), very weak
354 correlations were found, indicating that digestibility of raw starch will not predict digestibility of cooked
355 rice starches. In addition, rice starch cooked in excess water behaved differently when in contact with
356 amylolytic enzymes compared to starch being cooked in limited water. Thus, digestibility of cooked
357 starch in excess water should not be used to assume the digestibility of cooked starch in limited water,
358 especially in the case of rice cooked in a rice cooker with limited water to rice ratio.
359
17
360
361 Table 2 Extent and rate of digestion of rice starches in uncooked, cooked in excess water and in limited water A,B
Uncooked Cooked in excess water Cooked in limited water

Starch samples
30 min 300 min K (10-3min-1) 30 min 300 min K (10-3min-1) 30 min 300 min K (min-1 x10-3)
d c
Q5 14.4 ± 1.8 83.0 ± 1.8 5.66 ± 0.13cd 41.8 ± 2.2b
87.5 ± 0.3a
21.21 ± 1.32ab 14.3 ± 0.2 bc
91.3 ± 0.3 f
5.86 ± 0.09b
Luong Quang 13.0 ± 0.1c 82.9 ± 1.2c 5.42 ± 0.11c 44.1 ± 0.4cde 91.3 ± 1.1b 23.22 ± 1.10bc 15.3 ± 1.2c 69.9 ± 0.6a 8.05 ± 0.48c

IR50404 7.2 ± 0.2b 72.7 ± 1.1b 3.55 ± 0.07b 45.1 ± 0.7def 87.6 ± 1.5a 23.62 ± 0.02cd 13.6 ± 0.4b 88.7 ± 1.1e 5.59 ± 0.08b
OM6976 5.4 ± 0.7a 71.9 ± 1.1b 3.55 ± 0.01b 39.2 ± 2.1a 90.8 ± 1.4b 19.89 ± 1.11a 9.5 ± 1.2a 81.1 ± 1.6c 4.40 ± 0.07a
OM4218 7.7 ± 0.7b 61.3 ± 0.4a 2.94 ± 0.04a 42.9 ± 1.2bc 90.3 ± 0.6b 23.30 ± 1.50bcd 21.5 ± 0.9d 75.9 ± 0.8b 11.86 ± 0.19d
Tam Xoan 7.9 ± 0.6b 72.3 ± 0.8b 3.52 ± 0.03b 43.2 ± 0.5bcd 90.6 ± 0.9b 23.85 ± 0.20cd 23.2 ± 0.9e 70.8 ± 0.5a 15.09 ± 0.41f
VD20 14.8 ± 0.7e 92.5 ± 1.2e 6.52 ± 0.07e 39.9 ± 0.8a 90.2 ± 1.3b 21.24 ± 0.23ab 25.0 ± 1.0f 96.1 ± 0.7h 8.80 ± 0.10c
Jasmine 16.0 ± 0.8ef 84.1 ± 0.8c 5.95 ± 0.04d 45.4 ± 1.0ef 87.5 ± 0.6a 25.68 ± 0.58de 36.3 ± 1.1h 90.2 ± 1.0ef 18.12 ± 1.10g
Bac Thom 18.0 ± 0.6fg 90.4 ± 1.6c 6.39 ± 0.12e 47.1 ± 1.0f 91.6 ± 1.1b 25.46 ± 3.03cde 20.6 ± 0.6d 83.7 ± 1.1d 8.75 ± 0.44c
Cai Hoa Vang 28.4 ± 0.1h 91.7 ± 0.1de 10.39 ± 0.29g 45.5 ± 0.5ef 90.9 ± 0.9b 26.84 ± 1.32e 21.4 ± 1.0d 93.8 ± 0.4g 8.42 ± 0.07c
White glutinous 23.3 ± 0.6g 84.8 ± 0.3c 10.02 ± 0.53f 44.8 ± 0.5cde 90.8 ± 1.1b 25.09 ± 0.23cde 29.2 ± 0.8g 97.2 ± 0.7h 12.72 ± 0.24e
A
362 Values are means ± standard deviation of triplicates
B
363 Values with different superscripts in each column are significantly different at p<0.05 determined using SPSS,Version 22
364
365
366
367
368
369
370
18
371 Undigested 1 hour digested 3 hours digested

B
Fig. 4 SEM images of Cai Hoa Vang (A), VD20 (B), OM4218 (C) and Luong Quang (D) starches in forms of undigested, digested for 1 hour and 3
hours at the same magnification of 3000X
19
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372 4 Conclusion
373 Starches with a wide range of amylose contents vary in swelling power, pasting and thermal
374 properties and supra-molecular structure. Amylose in rice starch seemed to be a factor limiting
375 the rate of digestion of the raw and the cooked starch in excess water. However, cooked starch in
376 limited water behaved differently to amylolysis compared to raw starch and cooked starch in

377 excess water. The cooking in excess water increased the rate of digestion up to 8 folds compared
378 to raw starch. The structural rearrangement of starches when being cooked in limited water may
379 act as a barrier for enzymatic digestion resulting in lower rate coefficients compared to those of
380 starches cooked in excess water. Amylose content, physicochemical properties and molecular
381 order of starch suitably explain the raw starch but not cooked starch digestibility. The
382 digestibility of raw rice starch and rice starch cooked in excess water are not appropriate for
383 predicting digestibility of cooked starches in limited water. Caution should be taken when
384 choosing rice varieties for preventing and managing type 2 diabetes based on digestibilities of
385 raw starch and cooked in excess water. Cooking conditions should be very carefully chosen to
386 predict the digestibility of cooked rice grains as they should reflect the domestic cooking
387 conditions.
388
389 References
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Published on 18 July 2016. Downloaded by Cornell University Library
Digestion of rice starches cooked in limited water is significantly different to raw or when
cooked in excess water
Physicochemical
properties Weak correlation
100 B
80
h y d ro ly s e d s ta rc h (% )

60
Isolated Cooked in 40
rice starch excess water

20
0
0 60 120 180 240 300
D ig e s t io n t im e ( m in )
1 0 0
A
8 0
h y d r o ly s e d s t a r c h (% )
6 0
4 0
Strong correlation
2 0
0
0 6 0 1 2 0 1 8 0 2 4 0 3 0 0
D ig e s t io n tim e (m in )
Cooked in 1 0 0
8 0
C
h y d r o ly s e d s t a r c h (% )
limited water 6 0
4 0
V D 2 0
6 9 7 6 J A S M IN E
L u o n g Q u a n g Q 5
2 0
O M 4 2 1 8 B a c T h o m
W H IT E G L U T IN O U S C a i H o a V a n g
IR 5 0 4 0 4 T a m X o a n
0
0 6 0 1 2 0 1 8 0 2 4 0 3 0 0
D ig e s t io n tim e (m in )
No correlation

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3 Tien Dat Huynh1, Ashok K Shrestha2 and Jayashree Arcot1*

Food & Function Accepted Manuscript

8 Authors’ email address: Tien Dat Huynh: tien.d.huynh@unsw.edu.au

9 Dr. Ashok Shrestha: A.Shrestha@uws.edu.au

10 Associate Professor Jayashree Arcot: j.arcot@unsw.edu.au

11 *Corresponding author: A/Prof. Jayashree Arcot, School of Chemical Engineering, The

13 9385 5966, Email: j.arcot@unsw.edu.au

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35 Starch, physicochemical, digestibility, cooking

39 is eaten as whole cooked grain, mostly by steaming or sometimes followed by stir-frying.

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47 rice 7, 8. Implications based on relationships between physicochemical properties and digestion of

56 in limited water conditions. In this paper, a comprehensive study on physicochemical properties

59 water was performed to clarify their relationship.

60 2 Materials and methods

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68 2.2 Starch isolation

78 were tested for physicochemical properties and in vitro digestion.

87 Scientific, Warriewood, Australia).

88 2.4 Rice starch thermal properties (DSC)

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94 Universal Analysis Software version 4.5.

96 2.5 X-ray diffraction (XRD) and relative crystallinity of rice starches

105 Systems, LLC, St. Petersburg, Russia).

106 2.6 Fourier Transform Infrared (FTIR) spectroscopy

112 ratios were calculated.

113 2.7 In vitro digestibility of rice starch

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138 same in vitro digestion procedure described above.

139 2.8 Microscopy

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146 accelerating voltage of 15-20 kV and a working distance of 10 mm.

148 2.9 Data fitting and statistical analysis

151 C = C∞ (1- e-Kt) (1)

158 SPSS version 22 (SPSS Inc.)

160 3 Results and discussion

161 3.1 Apparent amylose content

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175 3.2 Swelling power of starch (SP)

190 non-waxy starches swell more freely.

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Swelling powers RVA parameters

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211 offset for clarity

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229 double helices of this starch.

230 3.5 Pasting properties of rice starch

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247 rice starch (150 RVU) in this study.

248 3.6 Thermal properties of rice starches

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270 lower their gelatinization enthalpies.

273 Data have been offset for clarity

274 3.7 In vitro digestion of rice starches

275 3.7.1 In vitro digestion of uncooked rice starches

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