Starch spherulites prepared by a combination of enzymatic and acid

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Food and Beverage Chemistry/Biochemistry

Starch spherulites prepared by a combination of enzymatic and acid hydrolysis of normal corn starch Yaqian Shang, Chen Chao, Jinglin Yu, Les Copeland, Shuo Wang, and Shujun Wang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01370 • Publication Date (Web): 04 Jun 2018 Downloaded from http://pubs.acs.org on June 4, 2018

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Journal of Agricultural and Food Chemistry

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Starch spherulites prepared by a combination of enzymatic and acid

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hydrolysis of normal corn starch

3 Yaqian Shangabc, Chen Chaoabc, Jinglin Yua, Les Copelandd, Shuo Wange*, Shujun Wangabc*

4 5

a

6

State Key Laboratory of Food Nutrition and Safety, Tianjin University of Science &

7

Technology, Tianjin 300457, China

8 9

b

Key Laboratory of Food Nutrition and Safety, Ministry of Education, Tianjin University of

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Science & Technology, Tianjin 300457, China

11 12

c

School of Food Engineering and Biotechnology, Tianjin University of Science & Technology,

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300457, China

14 15

d

The University of Sydney, Sydney Institute of Agriculture, School of Life and

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Environmental Sciences, NSW Australia 2006

17 18

e Tianjin Key Laboratory of Food Science and Human Health, School of Medicine, Nankai

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University, Tianjin, 300071, China

20 21

* Corresponding authors: Dr. Shuo Wang or Dr. Shujun Wang

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Mailing address: No 29, 13th Avenue, Tianjin Economic and Developmental Area (TEDA), Tianjin

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300457, China

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Phone: 86-22-60912486

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E-mail address: [email protected] or [email protected]

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ACS Paragon Plus Environment


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ABSTRACT：This paper describes a new method to prepare spherulites from normal

28

corn starch by a combination of enzymatic (mixtures of α-amylase and

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amyloglucosidase) and acid hydrolysis followed by recrystallization of the hydrolyzed

30

products. The resulting spherulites contained a higher proportion of chains with

31

degree of polymerization (DP) 6-12 and a lower proportion of chains with DP 25-36,

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compared with native starch. The spherulites had an even particle size of about 2 µm

33

and a typical B-type crystallinity. The amounts of long- and short-range molecular

34

order of double helices in starch spherulites were larger, but the quality of starch

35

crystallites was poorer, compared with native starch. This study showed an efficient

36

method for preparing starch spherulites with uniform granule morphology and small

37

particle size from normal corn starch. The ratios of α-amylase and amyloglucosidase

38

in enzymatic hydrolysis had little effect on the structure of the starch spherulites.

39 40 41

Keywords: corn starch, starch spherulites, enzymatic hydrolysis, acid hydrolysis,

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freezing-thawing, molecular order.

43 44 45 46 47 48 2


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INTRODUCTION

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Microcrystalline starch is a starch-derived product with high degree of crystallinity

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and small particle size, typically less than 10 µm, but preferably less than 6 µm.1

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Microcrystalline starch products have many food applications including as emulsifier,

53

thermoplastic reinforcer, fat substitutes, and stabilizers in frozen foods to control ice

54

crystalline formation.1-4 Native starch from amaranth grain is a unique resource of

55

natural microcrystalline starch because of its very small starch granules of only 1-3

56

µm in diameter. 5 Microcrystalline starches are often prepared by hydrolysis of starch

57

using acid and/or enzymes below the gelatinization temperature.6 Microcrystalline

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starch spherulites (or spherocrystalline starch) have been prepared by recrystallization

59

of heated solutions of amylose-containing starch, purified amylose solutions,7-9

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acid-hydrolyzed starch,11,12 or linear short amylose chains prepared by debranching of

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waxy starches.14-19

62 63

Starch spherulites have spherical semi-crystalline form and exhibit a specific

64

birefringence when observed under a polarized light microscope.10 Helbert et al.11

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prepared A-type amylose spherulites by mixing ethanol with hot aqueous solutions of

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short chain amylodextrin (DP=15), followed by slow cooling to 4 oC. The particle size

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of the amylose spherulites obtained was about 10 µm. Ring et al.12 prepared B-type

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spherulites with smooth surfaces and particle diameter of 10-15 µm by cooling 5-20%

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(w/w) aqueous solutions of short chain amylodextrin (DP=22) to 2 oC. A-type

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spherulites were obtained when aqueous solutions containing 30% (w/w) ethanol were 3



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used. In these studies,11,12 short chain amylodextrin was obtained by extensive

72

hydrolysis of native potato starch in 2.2 M HCl at 35 oC for 35 days, with the yield

73

being very low (~5%). Fanta et al.13 prepared different spherocrystalline particles of

74

varied size and morphology by slowly cooling dilute, jet-cooked solutions of various

75

cereal starches. Starch spherulites were also prepared by melting and crystallization of

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linear short-chain amylose that was obtained by enzymatic debranching of waxy

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maize starch. Cai et al.14-17 prepared A- and B-type starch spherulites with high

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crystallinity by enzymatic debranching of waxy starches using isoamylase, followed

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by melting and recrystallization of obtained short-chain amylose. The size of

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spherulites formed at low temperature (4 and 25 oC) was larger (5-10 µm) than those

81

crystallized at 50 oC (1-5 µm). In other studies,18,

82

prepared by a similar method, in which pullulanase was used to debranch waxy maize

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starch. While these preparation methods are simple and the yield is high, most of

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these studies used waxy maize starch as starting materials and the particle size was

85

often larger than 5 µm. Little information is available on the preparation of starch

86

spherulites using normal or high-amylose starches.

19

starch nanospherulites were

87 88

In previous studies, starch spherulites were often prepared from short-chain

89

amylodextrin obtained by acid hydrolysis of starch. However, acid hydrolysis of

90

starch is time-consuming and the yield of amylodextrin is very low (~5%).11,12

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Recently, enzymatic pretreatment using α-amylase, β-amylase or glucoamylase

92

followed by acid hydrolysis has been used to prepare starch nanocrystals from waxy 4


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maize starch, with increased yield and reduced preparation time.20 According to this

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study, we speculated that enzymatic pretreatment followed by acid hydrolysis can be

95

used to prepare starch spherulites with increased yield and reduced preparation time.

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In the present study, we aimed to develop a new method to prepare spherulites from

97

normal (i.e., non-waxy) corn starch by a combination of enzymatic (mixtures of

98

α-amylase and amyloglucosidase) and acidic hydrolysis followed by recrystallization

99

of the dissolved product. The starch spherulites obtained were characterized

100

comprehensively by high performance anion exchange chromatography (HPAEC),

101

light microscopy (LM), scanning electron microscopy (SEM), X-ray diffraction

102

(XRD),

103

spectroscopy, laser confocal micro-raman (LCM-Raman) spectroscopy, and

104

differential scanning calorimetry (DSC). To the best of our knowledge, this is the first

105

study to prepare starch spherulites from normal corn starch with smaller particle size

106

and uniform particle morphology.

attenuated

total

reflectance-fourier

transform

infrared

(ATR-FTIR)

107 108

EXPERIMENTAL SECTION

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Materials. Corn starch (10.2% moisture, 27.1% amylose), α-amylase (EC 3.2.1.1, 16

110

U/mg) from porcine pancreas, and amyloglucosidase (EC 3.2.1.3, 3260 U/mL) were

111

purchased from Sigma Aldrich Chemical Corporation (Shanghai, China). Pullulanase

112

(1000 ASPU/g) was purchased from Yuanye Biotechnology Co. Ltd (Shanghai,

113

China). Disodium hydrogen phosphate dodecahydrate (Na2HPO4·12H2O), citric acid

114

(C6H8O7·H2O), sulfuric acid (96~99 wt%), and ethanol (>99%) were all purchased 5



115

from Sinopharm Chemical Reagent Co. Ltd (China).

116 117

Enzymatic Hydrolysis of Starch. Enzymatic hydrolysis of starch was conducted as

118

described by Zhang et al.21 with modifications as follows using ratios of α-amylase to

119

amyloglucosidase activities of: 1:0, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6,

120

0:1, respectively. Starch (35 g, dry basis) was weighed accurately into a 500 mL

121

beaker, and then the α-amylase and amyloglucosidase mixtures (total activities were

122

12220 U) were added into the beaker. Subsequently, 240 mL of the citric

123

acid–disodium hydrogen phosphate buffer solution (0.2 M, pH 5.0) was added. The

124

enzymatic reaction was performed at 50 oC for 12 h. After hydrolysis, the precipitate

125

was washed with distilled water to neutral pH, and absolute ethanol was added to

126

dehydrate the starch products. The enzymatically-treated starch was dried under

127

gentle air stream at room temperature.

128 129

Acid Hydrolysis of Enzymatically-treated Starch. The acid hydrolysis of

130

enzymatically-treated starches were conducted as described by Angellier et al.22 In

131

brief, 147 g of enzymatically-treated starch was suspended in 1 L of 3.16 M H2SO4

132

and incubated in a water bath at 40 oC for 2 days under magnetic stirring at 100 rpm.

133

The final suspensions were washed to neutral pH with deionised water by successive

134

resuspension and centrifugation, and then freeze-dried to obtain starch dextrins.

135 136

Preparation of Starch Spherulites. The starch dextrins were dispersed in deiniosed 6


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water (5% solid concentration) and heated in a boiling water bath with continuous

138

stirring for 30 min. The hot solution was centrifuged at 1000g for 15 min to remove

139

the undissolved material, and the clear supernatant was cooled to room temperature

140

and immediately put into a freezer at -20 oC and left overnight. The frozen samples

141

were thawed slowly at room temperature, and the precipitate was washed with

142

distilled water three times and dried under a fume hood at room temperature. The

143

samples obtained were referred to as starch spherulites in the subsequent analysis.

144 145

High Performance Anion Exchange Chromatography (HPAEC). The chain length

146

distribution of starch spherulites was analyzed using a previous method with some

147

modifications23,

148

Corporation, Sunnyvale, CA, USA) with a pulsed amperometric detector (PAD).

149

Samples (18 mg) were dissolved in 900 µL 100% DMSO with constant stirring

150

overnight. The solution was diluted with 4.5 mL Milli-Q water and 600 µL 0.1 M

151

sodium acetate buffer (pH 5.5), and then 2 µL pullulanase (1000 ASPU/mL) was

152

added. The debranching reaction was conducted at 58 oC with slow stirring for 24 h.

153

The debranched samples were heated in a boiling water bath at 100 °C for 10 min to

154

inactivate the enzyme. The samples were cooled to room temperature and passed

155

through 0.22 µm nylon syringe filters and injected into the HPAEC system. The

156

debranched amylopectin chains were separated on a Dionex CarboPacTM PA200

157

column at 30 oC at a flow rate of 0.5 mL/min with gradient elution: 43% deionized

158

water (eluent A), 50% 200 mM NaOH (eluent B) and 7% 1 M NaOAc (eluent C) from

24

on the HPAEC system (HPAEC-PAD, ICS-5000+, Dionex

7



159

0 to 30 min, then 20 % eluent A, 50% eluent B and 30% eluent C from 30 to 50 min,

160

followed by 43% eluent A, 50% eluent B and 7% eluent C (the starting mixture) from

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50 to 60 min. The weight fractions of different chain lengths were quantified based on

162

the area of peaks.

163 164

Scanning Electron Microscopy. The morphology of starch samples was imaged

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using a JMS-IT300LV scanning electron microscope (JEOL, Japan). Samples were

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mounted on aluminum stubs using double-sided carbon adhesive tapes and

167

sputter-coated with gold. An accelerating voltage of 5 kV was used during imaging.

168 169

Light Microscopy. A light microscope (DM-400M-LED, Leica, Germany) was used

170

to observe the birefringence of native starch and starch spherulites. Approximately 10

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mg of each sample was weighed into a plastic tubes and 1 mL of deionised water was

172

added to suspend the sample. One drop of suspension was applied onto a microscope

173

slide, covered with a coverslip, and dried in a horizontal position for 5 min. A

174

polarized light mode was used for imaging.

175 176

X-ray Diffraction. X-ray diffraction analysis was performed using an X-ray

177

diffractometer (D8 Advance, Bruker, Germany) operating at 40 kV and 40 mA.

178

Samples were equilibrated over a saturated NaCl solution at room temperature for one

179

week before analysis. The X-ray diffraction patterns were measured from 4 to 35o (2θ)

180

and at a scanning speed of 2 o/min and a step size of 0.02 o. The relative crystallinity 8


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was quantitatively estimated using TOPAS 5.0 (Bruker, Germany).

182 183

Attenuated

Total

Reflectance-Fourier

Transform

Infrared

(ATR-FTIR)

184

Spectroscopy. The ATR-FTIR spectra of starch samples were obtained using a

185

Thermo Scientific Nicolet IS50 spectrometer (Thermo Fisher Scientific, USA). Starch

186

(150 mg) was weighed accurately and pressed into a transparent sheet and scanned

187

from 4000 to 400 cm−1. The spectra were obtained at a resolution of 4 cm−1 with an

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accumulation of 64 scans against air as the background. The full FTIR spectra were

189

baseline-corrected automatically by using OMNIC 6.2 before the spectra from 1200 to

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800 cm−1 were deconvoluted with a half band width of 19 cm−1 and an enhancement

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factor of 1.9. The ratio of absorbances at 1047/1022 cm−1 was used to estimate the

192

short-range ordered structure of starch.25

193 194

Laser Confocal Micro-Raman (LCM-Raman) Spectroscopy. The LCM-Raman

195

spectra of starch samples were obtained using a Renishaw Invia Raman microscope

196

system (Renishaw, Gloucestershire, United Kingdom) equipped with a Leica

197

microscope (Leica Biosystems, Wetzlar, Germany), and a 785 nm green diode laser

198

source was used. Spectra were taken from at least five different positions of each

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sample in the range of 3200~100 cm−1, with a resolution of approximately 7 cm−1.

200

The full width at half maximum (FWHM) of the band at 480 cm−1 was calculated

201

using the software of WIRE 2.0 to characterize the short-range ordered structure in

202

starch.26, 27 9



203 204

Differential Scanning Calorimetry (DSC). Differential Scanning Calorimetry (DSC)

205

measurements were performed using a differential scanning calorimeter (200 F3,

206

Netzsch, Germany) equipped with a thermal analysis data station. Starch (3 mg) was

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weighed accurately into an aluminum sample pan. Distilled water was added with a

208

pipette to obtain a starch: water ratio of 1:3 (w/w) in the DSC pans. The pans were

209

sealed and allowed to stand overnight at room temperature before analysis.28 The

210

samples were heated from 20 to 100 oC at a heating rate of 10 oC/min. An empty

211

aluminum pan was used as the reference. The onset (To), peak (Tp), conclusion (Tc)

212

temperatures and enthalpy change of gelatinization (△H) were obtained through data

213

recording software.

214 215

Statistical Analysis. All of the experiments were performed at least in triplicate,

216

except in the case of XRD, for which only one measurement was made. The data were

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analyzed using one-way analysis of variance (ANOVA) and were reported as mean

218

values and standard deviations by using the SPSS 19.0 Statistical Software Program

219

(SPSS, Inc. Chicago, IL, U.S.A.). The differences were considered at a significant

220

level of 95% (p < 0.05)

221 222

RESULTS AND DISCUSSION

223

Chain Length Distribution of Starch Spherulites

224

As all starch spherulites presented similar chain length distribution profiles, only 10


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those for native starch and starch spherulites prepared using one of the enzyme

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mixtures of α-amylase:amyloglucosidase (ratio of 6:1) is shown (Figure 1). The

227

proportions of each branch chain of amylopectin are summarized in Table 1. Branch

228

chains of amylopectin can be classified into four categories: A chain (DP 6-12), B1

229

chains (DP 13-24), B2 chains (DP 25-36), and B3+ chains (DP ≥ 37).29 The

230

proportions of A, B1, B2 and B3+ chains of amylopectin in native corn starch were

231

23.0, 44.9, 15.8 and 16.4%, respectively. Starch spherulites presented different chain

232

length distribution profiles compared to those of native starch. The most noticeable

233

feature was that all starch spherulites had a low proportion of very short chains of DP

234

2~5, accounting for about 5.0~10.0% of the total chains. Pullulanase, which was used

235

for debranching in our analysis, can hydrolyze the α-1,6 glucosidic linkages of

236

smaller branch chains, thus leading to the formation of maltose, maltotriose and other

237

small linear oligosaccharides.28,29 The proportions of A, B1, B2 and B3+ chains of

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starch spherulites varied slightly with the enzymatic hydrolysis conditions. Compared

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with native starch, all starch spherulites presented a higher proportion of chains with

240

DP 6-12 and a lower proportion of chains with DP 25-36. There were small

241

differences in the fractions of DP 13-24 and DP ≥ 37 between starch spherulites and

242

native starch. The increase in proportion of chains with DP 6-12 was attributed to the

243

cleavage of amylose or long branch chains of amylopectin by enzymes and acid.

244 245

A strong shoulder peak at DP 6~24, observed in all starch spherulites (Figure 1-B), is

246

assumed to originate mainly from external A chains and singly branched B1 chains, 11



247

which comprise double helices and are considered to be more resistant to attack by

248

enzymes or acid.30

249 250

Morphology of Starch Spherulites

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Figure 2 shows the SEM and LM (normal and polarized light) images of native corn

252

starch and starch spherulites. Native corn starch granules had rough surfaces and

253

irregular shapes with diameters of 5-35 µm (Figure 2-A). Starch spherulites, prepared

254

under various conditions, presented similar particle morphology and size (Figure 2-B,

255

2-C and 2-D). The ratio of α-amylase and amyloglucosidase used for the enzymatic

256

hydrolysis had little effect on particle morphology of the starch spherulites (although

257

the yield decreased as the amount of amyloglucosidase in the mixture increased) and

258

hence only a representative selection of images is shown. The spherulites had a

259

spherical shape with a much smaller particle size of 2 µm compared with native starch.

260

The particles presented a smoother and denser surface, although some particles

261

showed small orifices (often referred to as uncrystallized holes) on the surface. Most

262

of the starch spherulites seemed to aggregate, which could be attributed to the uneven

263

and imperfect crystallization of a wide range of short linear or branched chains during

264

the freezing and thawing processes. Starch spherulites obtained in the present study

265

had smaller and more uniform particles than those reported in previous studies. 14, 31, 32

266 267

Native starch granules displayed obvious birefringent patterns with the characteristic

268

Maltese cross, which were absent in the starch spherulites. These observations 12


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indicated that the starch spherulites had lost the concentric arrangement of crystalline

270

regions that give rise to birefringence. Similar observations were also reported for

271

starch spherulites prepared from waxy maize starch using enzymatic hydrolysis

272

followed by recrystallization by Cai et al.14 Under normal light microscope, mostly

273

aggregated particles were observed.

274 275

Long-range Ordered Structure of Starch Spherulites Determined by XRD

276

Figure 3 shows the diffraction patterns of native starch and starch spherulites, and the

277

values for relative crystallinity are summarized in Table 2. Native corn starch

278

exhibited a typical A-type diffraction pattern. 33 In contrast, the starch spherulites

279

displayed the typical B-type diffraction patterns, 33 with four clear diffraction peaks at

280

5.6°, 17.1°, 22.1°, 24.1° (2θ) and an overlapping peak at 14 and 15° (2θ). All of the

281

spherulites had much higher relative crystallinity (from 39.2% to 42.9%) than the

282

native starch (27.1%). Pretreatment of starch using different ratios of α-amylase and

283

amyloglucosidase had little effect on the relative crystallinity of starch spherulites,

284

suggesting that acid hydrolysis rather than enzymatic pretreatment had the main role

285

in hydrolyzing starch to the dextrins of similar chain length distribution.

286 287

Previous studies have shown that for crystallization of amylose chains, shorter chain

288

length (CL), higher concentration, and higher crystallization temperature favored the

289

formation of A-type crystallites, and vice versa for the B-type crystallization

290

structure.16, 17, 31 According to Gidley and Bulpin,34 malto-oligomer chains of DP < 10 13



291

in aqueous solution do not form double helices, DP 10~12 gives an A-type X-ray

292

diffraction pattern, and DP ≥ 13 gives a B-type pattern. The results of the present

293

study are in agreement with these findings, as seen by the low concentration of

294

dextrins (5% solids), low crystallization temperature (-20 oC) and a large proportion

295

of longer chains (CL > 12) inducing the formation of the B-type crystalline

296

polymorph.

297 298

Short-range Ordered Structure of Starch Spherulites Determined by ATR-FTIR

299

and LCM-Raman Spectroscopy

300

ATR-FTIR is a technique that can examine the structure of starch granules from the

301

surface to a depth of about 2-3 µm.35 The bands at 1047 and 1022 cm−1 are considered

302

to be associated with crystalline and amorphous regions, respectively.36, 37 The ratio of

303

absorbances at 1047/1022 cm−1 is used to characterize the short-range molecular order

304

of double helices, with higher absorbance ratios indicating a greater degree of

305

molecular order of double helices in starch.38 The ratio of absorbances at 1047/1022

306

cm−1 of the starch spherulites (Table 2) were increased significantly over that of native

307

starch, consistent with greater short-range molecular order in the spherulites. Again,

308

there were no significant differences in the absorbance ratios between the spherulites

309

prepared using different ratios of the amylolytic enzymes, providing further evidence

310

that this was not a key factor for the form of the starch spherulites.

311 312

Raman spectroscopy is also used to characterize short-range ordered structural order 14


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in starch, giving rise to five characteristic bands at 480, 865, 943, 1264 and 2900 cm−1,

314

which are related to δ (CH2), νs (C1-O-C4), νs (C1-O-C5), skeletal (C-C-O), and ν

315

(C-H) modes, respectively.39 The bands at 480 cm−1 and 2900 cm−1 are sensitive to

316

changes in structural order of starch,39, 40 with a lower value of the full width at half

317

maximum (FWHM) of the band at 480 cm−1 being associated with higher molecular

318

order.39 The FWHM of the band at 480 cm-1 was 15.70 and 14.63 to 14.91 for native

319

starch and starch spherulites, respectively (Table 2), indicating the greater short-range

320

structural order of starch spherulites compared with native starch. The similarity of

321

FWHM for the spherulites is consistent with there being no obvious differences in

322

their degree of molecular order.

323 324

Thermal Properties of Starch Spherulites

325

Thermograms of native starch and starch spherulites are shown in Figure 4, and the

326

onset, peak, conclusion temperature (To, Tp, and Tc, respectively) and enthalpy change

327

(△H) are summarized in Table 3. Native starch presented a typical narrow

328

gelatinization endotherm with To, Tp and Tc of 65.5, 70.9 and 76.2 o C, respectively.

329

The enthalpy change and gelatinization temperature range were 10.8 J/g and 10.7 oC,

330

respectively. The DSC traces of the starch spherulites were all similar, with a single

331

thermal transition at lower temperature but over a broader range from 16.9 to 18.8 oC.

332

The enthalpy change of starch spherulites ranged from 18.4 to 21.7 J/g, which were

333

much higher than that of native starch (10.8 J/g). The increased enthalpy change and

334

decreased thermal transition temperatures for starch spherulites indicated that the 15



335

quantity of starch crystallites increased but the quality of starch crystallites decreased.

336

Thus compared with native starch, the spherulites contained a higher proportion of

337

crystallites, but the degree of crystallite perfection was lower. The broader thermal

338

transition was indicative of the greater heterogeneity of starch crystallites formed in

339

starch spherulites, consistent with the broader chain length distribution of the

340

spherulites.

341 342

Mechanism of Formation of Starch Spherulites

343

Taking all of the above results into account, we propose a mechanism for the

344

formation of starch spherulites as represented schematically in Figure 5. Native

345

granules of non-waxy starch (Figure 5-A) are considered to have an amorphous core

346

containing mostly amylose and disordered amylopectin branch chains, which is

347

surrounded by a concentric pattern of alternating semi-crystalline and amorphous

348

growth rings (Figure 5-a).41 Enzymatic hydrolysis formed pores and channels from

349

the surface to the interior (Figure 5-B and 5-b), which facilitated the penetration of

350

acid towards the less-organized regions in the granule. Then, after acid hydrolysis, the

351

starch granules were disintegrated into small fragments containing unbranched and

352

branched chains (Figure 5-C) which remained associated due to chain entanglement.

353

With dissolution the chains dissociated and on recrystallization they self-assembled to

354

form starch spherulites (Figure 5-D). Due to the heterogeneity of dextrins produced

355

by enzymatic and acidic hydrolysis (Figure 5-c), the resulting starch spherulites had

356

imperfect structural features, as shown by the loss of birefringence and the presence 16


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of orifices on the particle surface (Figure 5-D). The loss of birefringence of the starch

358

spherulites indicated that the double helices remaining were not orientated radially,

359

and the crystallites were stacked irregularly from the center to the periphery (Figure

360

5-d).

361 362

In summary, a new method was developed to prepare starch spherulites with uniform

363

particle morphology and small particle size. The spherulites had a high proportion of

364

short branched and unbranched chains of DP 10 to 20, and an even particle size of 2

365

µm. The spherulites had typical B-type crystallinity, with larger amounts of long- and

366

short-range molecular order of double helices than native starch, but less perfect

367

crystallites. Different enzymatic pretreatments had little effect on the morphology and

368

ordered structures of the starch spherulites.

369 370

FUNDING

371

The authors gratefully acknowledge the financial support from the National Natural

372

Science Foundation of China (31522043) and Tianjin Natural Science Foundation for

373

Distinguished Young Scholar (17JCJQJC45600).

374 375

NOTES

376

The authors declare no competing financial interest.

377 378 17



379

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380

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486

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1564-1580.

492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 23



511 512

Table 1. Chain length distribution of amylopectin of native starch and starch spherulites. Branch chain length distribution of amylopectin (%) Samples DP 2-5 DP 6-12 DP 13-24 DP 25-36 DP≥37 A B C D E F G H I J K L M N

513 514 515 516

Page 24 of 33

0.0±0.0a 10.3±0.5e 6.1±0.3cd 6.1±0.5cd 5.7±0.5bcd 5.3±0.2bc 5.8±0.5bcd 6.3±0.5cd 6.7±0.4d 5.3±0.5bc 4.7±0.6b 6.3±0.5cd 5.8±0.7bcd 5.8±0.4bcd

23.0±0.0a 30.0±2.0c 29.8±1.8c 26.5±0.4abcd 26.8±0.5bcd 26.1±2.2abc 25.9±0.9ab 25.6±1.6abc 26.5±0.6abcd 28.2±0.0bcd 27.3±1.7bcd 26.7±1.0bcd 28.9±2.4cd 25.5±1.1abc

44.9±0.3cd 36.6±3.6a 49.0±1.6d 43.6±0.0bc 42.7±0.6bc 43.9±2.0bc 44.0±0.1bc 45.4±0.6cd 40.1±2.1ab 43.1±1.0bc 42.5±3.3bc 44.3±0.4bc 42.8±3.4bc 45.7±0.6cd

15.8±0.5a 8.6±0.1b 6.8±1.3b 7.3±2.2b 7.3±1.8b 7.3±1.7b 8.4±2.6b 8.3±2.6b 7.5±1.3b 6.3±1.0b 6.4±1.0b 6.9±2.4b 6.8±1.6b 9.2±0.3b

16.4±0.2ab 14.5±1.3a 15.0±3.7a 16.5±1.4ab 17.6±1.4ab 17.4±1.6ab 16.9±1.3ab 14.3±1.1a 19.1±1.0b 17.1±1.5ab 19.2±1.9b 15.9±1.3ab 15.7±1.9ab 13.9±0.5a

Values are means ± SD. The different lowercase letters represent significant differences between the data in the same column (p < 0.05). A : native starch, B-N: starch spherulites prepared using enzyme mixtures at activity ratio of α-amylase : amyloglucosidase =1:0, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 0:1 respectively.

517 518 519 520 521 522 523 524 525 526 527 24


Page 25 of 33


528 529

Table 2. The ratios of 1047/1022 cm−1, FWHMs of the band at 480 cm−1 and relative crystallinity of native starch and starch spherulites. Samples IR ratios of Relative FWHM -1 absorbances at at 480cm crystallinity（%） 1047/1022(cm-1) 0.618±0.013a A 15.70±0.13a 27.1 0.692±0.018b B 14.86±0.13b 41.6 0.684±0.019b C 14.85±1.05b 42.2 0.694±0.019b D 14.75±0.18b 40.7 0.691±0.010b E 14.75±0.67b 42.7 0.685±0.037b F 14.68±0.20b 41.7 0.682±0.010b G 14.64±0.14b 40.9 0.693±0.012b H 14.83±0.18b 43.9 0.692±0.014b I 14.68±0.11b 41.2 0.689±0.012b J 14.91±0.28b 40.6 0.695±0.023b K 14.84±1.18b 39.7 0.693±0.013b L 14.82±0.20b 39.2 0.694±0.014b M 14.80±0.88b 42.9 0.685±0.020b N 14.63±0.08b 40.3

530 531 532 533

Values are means ± SD. The different lowercase letters represent significant differences between the data in the same column (p < 0.05). A: native starch, B-N: starch spherulites prepared using enzyme mixtures at activity ratio of α-amylase : amyloglucosidase =1:0, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 0:1 respectively.

534 535 536 537 538 539 540 541 542 543 544 25



545

Table 3. Thermal properties of native starch and starch spherulites. Samples To（oC） △H（J/g） Tp（oC） Tc（oC） A B C D E F G H I J K L M N

546 547 548 549

65.5±0.2a 50.9±0.2bc 51.2±0.2cde 51.7±0.3ef 51.4±0.3de 52.0±0.2f 51.6±0.5ef 51.1±0.2cd 51.2±0.2cde 51.4±0.1de 50.8±0.2bc 51.6±0.2def 50.6±0.0b 50.8±0.2bc

70.9±0.1a 59.9±0.1cd 60.0±0.1d 60.4±0.2e 60.5±0.1e 60.4±0.1e 59.3±0.2b 59.3±0.3b 59.3±0.3b 59.6±0.2bc 59.8±0.2cd 59.6±0.2bc 59.6±0.1bc 59.4±0.2b

76.2±0.3a 69.1±0.4de 69.4±0.3e 69.9±0.1fg 70.1±0.1g 69.6±0.2ef 68.3±0.3bcd 68.3±0.3b 68.5±0.6bc 68.2±0.1b 69.0±0.3cde 68.7±0.3bcd 69.4±0.4e 68.4±0.1b

10.8±0.1a 21.7±0.2f 18.7±0.1bc 21.2±0.2ef 20.9±0.8e 21.0±0.5ef 19.8±0.5bc 19.8±0.5d 18.4±0.8b 19.4±0.4cd 18.9±0.5bc 19.9±0.7d 20.9±0.1ef 18.6±0.7bc

Page 26 of 33

△T（oC） 10.7±0.2a 18.2±0.2de 18.2±0.5de 18.2±0.2f 18.7±0.3f 17.6±0.3cd 17.2±0.1bc 17.2±0.1bc 17.3±0.5bc 16.9±0.1b 18.2±0.3de 18.8±0.4bc 18.6±0.4f 17.6±0.3cd

Values are means ± SD. The different lowercase letters represent significant differences between the data in the same column (p < 0.05). A : native starch, B-N: starch spherulites prepared using enzyme mixtures at activity ratio of α-amylase : amyloglucosidase =1:0, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 0:1 respectively.

550 551 552 553 554 555 556 557 558 559 560 561 26


Page 27 of 33


562

Figure captions

563

Figure 1. Chain length distribution of amylopectin of native starch (A) and starch

564

spherulites obtained at ratio of α-amylase to amyloglucosidase activities of 6:1 (B).

565 566

Figure 2. Representative scanning electron microscopy (A-D) and light microscopy

567

images under normal (A1-D1) and polarized light modes (A2-D2) of native starch (A)

568

and starch spherulites (B-D). B-D: starch spherulites prepared using enzyme mixtures

569

at activity ratio of α-amylase : amyloglucosidase =1:0, 1:1, 0:1 respectively.

570 571

Figure 3. Wide-angle X-ray diffractgrams of native starch and starch spherulites. A :

572

native starch, B-N: starch spherulites prepared using enzyme mixtures at activity ratio

573

of α-amylase : amyloglucosidase =1:0, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5,

574

1:6, 0:1 respectively.

575 576

Figure 4. Thermal properties of native starch and starch spherulites. A : native starch,

577

B-N: starch spherulites prepared using enzyme mixtures at activity ratio of α-amylase :

578

amyloglucosidase =1:0, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 0:1

579

respectively.

580 581

Figure 5. The preparation process from native corn starch to starch spherulites. A, a :

582

native starch, B, b : porous starch, C, c : dextrin, D, d : starch spherulites.

583 584 585 586 587 588 589 590

27



591

Page 28 of 33

Figure 1 5

A

%Distribution

4 3 2 1 0

0

1 0

2 0

3 0

592

4 0 5 0 D P

6 0

7 0

8 0

6

B

%Distribution

5 4 3 2 1 0

0

1 0

2 0

3 0

4 0 5 0 D P

593 594 595 596 597 598 599 600 601 602 603 604 605 606 28


6 0

7 0

8 0

Page 29 of 33


607

Figure 2 A

A1

A2

B

B1

B2

C

C1

C1

D

D1

D2

608

609

610

611 612 613 614 615 616 29



617

Page 30 of 33

Figure 3

25000 N M L K J

Diffraction intesity(PSD)

20000

15000

I H G F E D C B A

10000

5000

0 5

618

10

15

20

25

30

2 Theta(°)

619 620 621 622 623 624 625 626 627 628 629 630 631 30


35

Page 31 of 33


632

Figure 4

8

N M L

DSC•flow

7

K J I H

6

G F E D C B

5

4

A 30

633

40

50

60

70

80

Temperature (ºC )

634 635 636 637 638 639 640 641 642 643 644 645 646 647 31


90


648

Page 32 of 33

Figure 5 A

B

C

D

649 650

(×3000)

(×5000)

(×10000)

651

b

a

d

c

652 653 654 655 656 32


(×3000)

Page 33 of 33


657

Table of Contents Graphic

658

659 660 661

33


Starch spherulites prepared by a combination of enzymatic and acid

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