Lamellar structure changes in rice starch during α-amylase hydrolysis: Effect of starch granule surface and channel proteins

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Food Bioscience 61 (2024) 104502

Available online 12 June 2024

Lamellar structure changes in rice starch during

-amylase hydrolysis:

Effect of starch granule surface and channel proteins

Xiaoning Liu

, Zekun Xu

, Xingxun Liu

, Chuangchuang Zhang

, Mengting Ma

Zhongquan Sui

, Harold Corke

Department of Food Science & Technology, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai, 200240, China

Shanghai Jiao Tong University Sichuan Research Institute, Chengdu, 610218, China

Lab of Food Soft Matter Structure and Advanced Manufacturing, College of Food Science and Engineering, Nanjing University of Finance and Economics, Nanjing,

210023, China

Biotechnology and Food Engineering Program, Guangdong Technion-Israel Institute of Technology, Shantou, 515063, China

Faculty of Biotechnology and Food Engineering, Technion-Israel Institute of Technology, Haifa, 3200003, Israel

ARTICLE INFO

Keywords:

Rice starch

Starch granule surface and channel proteins

(GSCP)

Starch granule channel proteins

-amylase hydrolysis

Lamellar structure

ABSTRACT

Starch granule surface and channel proteins (GSCP) or starch granule channel proteins (GCP) were respectively

removed from waxy and non-waxy rice starches. The effects of

-amylase hydrolysis on the lamellar structure

changes of rice starches were examined by small-angle X-ray scattering (SAXS). SAXS revealed that the scattering

peaks of hydrolyzed starch granules were broadened. The result was also evidenced by the decreased peak areas

(A) and increased peak width at half-maximum (W). The

-amylase hydrolysis of starches could induce a

transformation from a mass to a surface fractal and increase the compactness of starches. Moreover, the thickness

of amorphous lamellae (d

) decreased and that of crystalline lamellae (d

) increased for starches after hydrolysis.

Hydrolyzed starches exhibited a higher proportion of short-branch chains (fa) and long-branch chains (fb3)

compared to their native form, indicating that

-amylase mainly attacked the long molecular chain of amylo-

pectin with branch points in the amorphous lamellae. Removal of GSCP accelerated and exacerbated the decrease

in d

and the increase in d

. Further, GSCP removal brought on the formation of less compact starch during long-

time hydrolysis. Overall, the results demonstrated that GSCP removal had signicant impacts on structural

changes during

-amylase hydrolysis from lamellar to molecular scales.

1. Introduction

The structure of native starch granules is organized at different

length scales, namely whole granule (

m), growth rings (~0.1

m),

lamellar structure (8–11 nm) and molecular scale (~0.1 nm) (P´

erez &

Bertoft, 2010; Tran et al., 2011). It is well known that native starch

granules consist of alternating amorphous and semi-crystalline growth

rings. Semi-crystalline growth rings are composed of repeats of alter-

nating amorphous and crystalline lamellae. Amorphous lamellae is

associated with branching points of amylopectin side chains, whereas

crystalline lamellae is formed by short-chains of amylopectin, individ-

ually arranged in double helices and encapsulated in small crystallites

(Witt et al., 2012). Furthermore, linear amylose molecules and possibly

less ordered amylopectin exist in an amorphous region within the native

granule (Fan et al., 2013; P´

erez & Bertoft, 2010).

Starch granule displays hierarchical structures in its native state, and

thus can be studied by small-angle X-ray scattering (SAXS) (Blazek &

Gilbert, 2011). SAXS has been used to study the changes in lamellar

structure of starch induced by enzyme hydrolysis (Blazek & Gilbert,

2010; Chanvrier et al., 2007; Lan et al., 2016; Lopez-Rubio et al., 2007).

Chanvrier et al. (2007) studied the lamellar structure of maize and

wheat starches by SAXS in the native and digested form and found that

digestion resulted in a reduction in lamellar peak intensity. Lopez-Rubio

et al. (2007) reported that amylolysis did not signicantly change the

lamellar peak in high amylose maize starch. Blazek and Gilbert (2010)

observed a gradual decrease in lamellar peak intensity and an increase in

low-q scattering for several starches during enzymatic hydrolysis. These

trends are more pronounced for type A starches than for type B starches.

The amorphous growth rings are preferentially digested, and enzymes

* Corresponding author. Department of Food Science & Technology, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai, 200240, China.

** Corresponding author. Shanghai Jiao Tong University Sichuan Research Institute, Chengdu, 610218, China.

E-mail addresses: mmt9497@sjtu.edu.cn (M. Ma), zsui@sjtu.edu.cn (Z. Sui).

Contents lists available at ScienceDirect

Food Bioscience

journal homepage: www.elsevier.com/locate/fbio

https://doi.org/10.1016/j.fbio.2024.104502

Received 15 March 2024; Received in revised form 18 May 2024; Accepted 3 June 2024

Food Bioscience 61 (2024) 104502

can penetrate and hydrolyze the amorphous region of semi-crystalline

growth rings. The thickness of lamellar structure of canna starch in-

creases in amorphous lamellae but decreases in crystalline lamellae after

-amylase treatment (Lan et al., 2016). It is inconsistent with previous

research that the thickness of semi-crystalline lamellae is 2–5 nm in

amorphous regions and 5–7 nm in crystalline regions, respectively (Le

Corre et al., 2010). Therefore, it is necessary to systematically investi-

gate the detailed information about lamellar structure of starch during

enzymatical hydrolysis.

The granule microstructure (pores/channels) and the nanostructure

of the growth-rings inuence the access of enzymes to their substrates,

determining the enzymatic digestibility (Blazek & Gilbert, 2010). Starch

granule-associated proteins are distributed on the surface, channels and

matrix of starch granules, and are respectively called granule

surface-proteins, granule channel-proteins (GCP) and granule

intrinsic-proteins (Kasarda et al., 2008). Extraction of starch granule

surface and channel proteins (GSCP) induces nano-scale surface

roughening, greater specic surface area and larger channel diameter

(pore size) in starch granules (Ma et al., 2021). Moreover, GSCP removal

increases the rate of

-amylase hydrolysis of rice starch and results in

larger pores, channels, and cavities at the granular level. However, at the

lamellar and molecular structure levels, the changes of rice starch after

removal of GSCP during

-amylase hydrolysis are still unknown.

In this study, rice starch granules after removal of GSCP or GCP

during

-amylase hydrolysis were analyzed by SAXS. We determined the

change of the lamellar structure during

-amylase hydrolysis by 1D

linear correlation and fractal analysis. The amylopectin ne structure

was also determined to investigate the structural changes at the mo-

lecular level, which may provide a more profound insight into the

changes in

-amylase hydrolyzed starch and the inuence of GSCP on

starch.

2. Materials and methods

2.1. Materials

BP602 (waxy rice) and JA166 (non-waxy rice) were provided by

Zhejiang University. Amylose contents(AACC, 2000; Method 61-03) on

a dry starch basis were 0.1% and 14%. Porcine pancreas

-amylase

(A3176) and phosphate buffer saline (PBS) were supplied by Sigma

Aldrich (St. Louis, MO). Sodium carbonate and ethanol were purchased

from Aladdin Reagent Co., Ltd. (Shanghai, China).

2.2. Removal of starch granule-associated proteins from starches

Starches were extracted from waxy and non-waxy rice to obtain WRS

and NRS and GCP or GSCP were extracted from starches (WRS-C, NRS-C

or WRS-S, NRS-S) followed the method of Zhan et al. (2020). In brief,

starch (70 g, d.b.) was thoroughly mixed with 200 mL buffer and the

suspension was magnetically stirred for 48 h. The buffer for extraction of

GCP contains 0.1% SDS, 0.2% mercaptoethanol, 50 mM Tris-HCl (pH 8)

and 10% glycerol, and 1.5% SDS, 2% mercaptoethanol, 50 mM Tris-HCl

(pH 8) and 10% glycerol for extraction of GSCP. After the centrifugation

(6000×g, 15 min), starch layer was resuspended in ethanol (80%, 500

mL) and ltered through a Buchner funnel. Then starches were dried at

room temperature to a constant weight. The protein content for WRS,

WRS-C, WRS-S and NRS, NRS-C, NRS-S and were 0.27%, 0.13%, 0.10%

and 0.54%, 0.32%, 0.20%, respectively.

2.3. Staining protein in rice starch with uorescamine

Starches (15 mg) were dispersed in 0.1% (w/v) uorescamine (0.3

mL) in acetonitrile and 0.15 mL of 0.1M borate buffer (pH 8.0) at 25 ◦C

for 1 h, then centrifuged at 10000×g for 5 min and washed ve times

with MilliQ water. The samples were suspended in 50% glycerol, then

observed using a Super-resolution Multiphoton Confocal Microscope

(TCS SP8 STED 3X, Leica, Wetzlar, Germany) with a 100 ×oil objective

lens according to the previous method (Naguleswaran et al., 2011).

Images were collected with a 10-mW argon ion laser at 15% power with

405 excitations.

2.4.

-amylase hydrolysis of starches

The

-amylase hydrolysis of starches followed the method of Ma

et al. (2020). The activity of porcine pancreatic

-amylase was measured

as 7.5 units/mL according to the method of Gonz´

alez et al. 2002. One

unit of the enzyme activity is dened as the amount that liberates 1.0 mg

of maltose from starch in 3 min at pH 6.9 and 20 ◦C. Samples (50 mg, d.

b) was mixed with 15 mL PBS (0.01 M, pH 7.2) in a tube, then mixed

with porcine pancreatic

-amylase solution (2 mL, 15 U). Mixed system

was incubated in a shaking water bath at 37 ◦C. The reaction was

stopped using ice-cold sodium carbonate solution (0.5 M), then samples

were collected at 0, 10, 20, 60, and 120 min. The residue was then

re-washed with ethanol were then freeze-dried after centrifugation to

obtain native and hydrolyzed starches.

2.4.1. Scanning election microscopy (SEM)

Hydrolyzed starches with 120 min were placed on a double-sided

carbon adhesive tape and coated with gold. Images were collected

using a super resolution eld emission scanning electron microscope

(ZEISS GeminiSEM 450, Oberkochen, Germany) at an accelerating

voltage of 3 kV.

2.5. Small-angle X-ray scattering (SAXS)

Synchrotron time-resolved SAXS measurements were conducted on

the BL19U2 beam line in Shanghai Synchrotron Radiation Facility

(SSRF, China) using the method of Xu et al. (2020). The suspension with

a ratio of starch: distilled water of 1:9 (w/w) was equilibrated for 24 h

before SAXS tests. The suspension was loaded into 2-mm-thick sample

cells which were covered with Kapton tape. Data was measured for 1 s.

The detailed parameters for SSRF testing and the data were background

subtracted and normalized followed the previous report (Liu et al.,

2017).

2.6. Analysis of SAXS data

The SAXS data was tted to a power-law function plus a Gaussian

peak (Eq. (1)) (Blazek & Gilbert, 2010):

I(q)=B+Pq−

+A̅̅̅̅̅̅̅̅

ln 2

√

W̅̅̅̅̅̅̅̅

√exp (−4ln 2(q−q0)2

W2)(1)

Where, B is the background; P is the power law pre-factor and

is the

power-law exponent; A is the Gaussian peak area, W (nm

−1

) is the full

width at half maximum of the peak, and q

(nm

−1

) is the peak center

position.

Abbreviations

GCP starch granule channel proteins

GSCP Starch granule surface and channel proteins

NRS non-waxy rice starch

NRS-GSCP non-waxy rice starch after removing GSCP

NRS-GCP non-waxy rice starch after removing GCP

WRS waxy rice starch

WRS- GSCP waxy rice starch after removing GSCP

WRS-GCP waxy rice starch after removing GCP

SAXS Small-angle X-ray scattering

X. Liu et al.

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