Slowing the Starch Digestion by Structural Modification through

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Food and Beverage Chemistry/Biochemistry

Slowing the Starch Digestion by Structural Modification through preparing Zein/Pectin Particle Stabilized Water-in-Water Emulsion Jia-Feng Chen, Jian Guo, Tao Zhang, Zhi-Li Wan, Juan Yang, and Xiao-Quan Yang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b05501 • Publication Date (Web): 06 Apr 2018 Downloaded from http://pubs.acs.org on April 6, 2018

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Journal of Agricultural and Food Chemistry

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Slowing the Starch Digestion by Structural Modification through

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preparing Zein/Pectin Particle Stabilized Water-in-Water Emulsion

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Jia-Feng Chen, Jian Guo *, Tao Zhang, Zhi-Li Wan, Juan Yang, and Xiao-Quan Yang

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Protein Research and Development Center, Guangdong Province Key Laboratory for

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Green Processing of Natural Products and Product Safety, National Engineering

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Laboratory of Wheat & Corn Further Processing, South China University of

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Technology, Guangzhou 510640, PR China

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Corresponding to: Jian Guo

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Protein Research and Development Center, School of Food Science and Technology,

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South China University of Technology, Guangzhou 510640, PR China

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Tel: +86 20 87114262

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Fax: +86 20 87114263

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E-mail address: [email protected]

ACS Paragon Plus Environment


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Abstract

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Slowing the digestion of starch is one of the dominant concerns in food industry. A

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colloidal structural modification strategy for solving this problem was proposed in this

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work. Due to thermodynamic incompatibility between two biopolymers, water/water

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emulsion of waxy corn starch (WCS) droplets dispersed in a continuous aqueous guar

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gum (GG) was prepared. And zein particles (ZPs), obtained by anti-solvent

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precipitation and pectin modification, were used as stabilizer. As the ratio of zein to

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pectin in the particles was 1:1, their wetting properties in the two polysaccharides

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were similar, which made them accumulate at the interface and cover the WCS-rich

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droplets. The analysis of digestibility curves indicated that a rapid (rate constant k1:

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0.145 min-1) and a slow phase (k2: 0.022 min-1) existed during WCS digestion. But

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only one slow phase (k2: 0.019 min-1) was found in the WCS/GG emulsion,

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suggesting that this structure was effective in slowing starch digestion.

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Keywords

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starch digestion, food structure design, water-in-water emulsion, zein particles

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Introduction

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Although people cook meals or prepare foods to meet the needs of satisfying

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hunger and pleasing the appetite, nutrition and health which is closely related to

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people’s living quality have always been the focus in food processing. Among the

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concerns, the sudden increase of postprandial glycemic level induced by the digestion

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of carbohydrates has been found to be associated with some health problems, such as

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diabetes, obesity and cardiovascular diseases.1,2 Reducing this fluctuation is conducive

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to weight control and chronic disease prevention.3

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Starch, which is contained in various staple foods, is one of the most common

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components in human diet. This polymeric carbohydrate provides energy, satiety and

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special texture perception for human beings. As it is broken down in the gastrointestinal

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tract, the released glucose is transferred into blood circulation, leading to an increase of

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blood sugar concentration.4 The rate and extent of starch hydrolysis are determinants of

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this metabolic response to a meal.5,6 In this context, the effects of origin, composition,

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structure, crystallinity, morphology, processing and cooking methods on starch

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digestibility has been well documented to predict the blood glucose response of

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starch.7-10 The glycemic index (GI) tables have been developed to provide a reference

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for people to choose foods.11-12 People consume a high-GI diet for years are at higher

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risk of developing chronic diseases. Especially those with diabetes should stay away

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from the high-GI foods for reason of health.

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Since people pay more attention to the taste of food, sacrificing the diversity and

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palatability of their diet might not be a preferable solution. Recently, the notion of food



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structure design has been applied in the preparation of model food,13-16 providing

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another perspective in developing healthier diet without quality loss. As for slowing

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starch hydrolysis, making its interaction with related degrading enzymes become

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difficult is a proven potential method. In this case, fabricating a protective layer coating

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on the surface of starch is a prerequisite. This analogous core-shell structure is often

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formed with the help of the templates in emulsion systems, which derive from the

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incompatibility of two phases and the accumulation of amphipathic molecules at the

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interface. Considering the edibility, operability of preparation and developing tendency

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of healthier diet, water-in-water (W/W) emulsion that is composed of two immiscible

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biopolymer liquids is more promising in synthesizing such structure, when compared to

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oil-water (O/W) system. Unfortunately, this fat-free emulsion has received less

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attention and limited applications due to its poor stability. By comparison, the

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interfacial tension between two phases is extremely low. And the interface is ill-defined

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and thicker.17-19 As a result, typical emulsifiers are hard to adsorb at W/W interfaces.

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The lack of protection for the droplets leads to rapid and irreversible macroscopic phase

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separation. In recent years, a wide variety of biopolymer-based particles have been

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used as stabilizing agents to obtain Pickering type O/W emulsions.20 Inspired by this,

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some food-grade particles have also been found to be capable of accumulating at W/W

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interfaces.21-25 In addition to gelation, this opens another way to fabricate food structure

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by means of stable W/W emulsion.

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In this work, we attempted to reduce the digestion rate and extent of gelatinized

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waxy corn starch (WCS) via creating specific colloidal structure. For this purpose, guar


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gum (GG), which had been reported to have inhibitory effect on the activity of

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α-amylase,26 was served as another incompatible polysaccharide to prepare

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food-grade W/W emulsion. Within this structure, starch droplets were embedded in GG

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liquid. Due to the amphiphilic structure as well as low solubility in both water and oil,

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zein particles (ZPs) have been reported to be effective particle stabilizers in various

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Pickering O/W emulsion systems.27-30 To modify their wettability at the interface,

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pectin was employed during their preparation. And the resulting particles were used as

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stabilizers in the W/W emulsions. Phase diagram of WCS/GG mixtures, wetting

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properties of ZPs, microstructure and stability of the obtained W/W emulsion are

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reported. More importantly, the effect of W/W emulsion structure on the hydrolysis

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kinetics of WCS were investigated.

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Materials and Methods

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Materials

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WCS was purchased from Qinhuangdao Lihua Starch Co. Ltd. (Hebei, China)

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Pectin and GG were purchased from Shanghai Toong Yeuan Food Technology Ltd.

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(Shanghai, China). Zein from corn and the digestive enzymes used in this work (Pepsin

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from porcine gastric mucosa, EC 3.4.23.1, 3200-4500 U/mg protein; Pancreatin from

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porcine pancreas, 8×USP specifications; Amyloglucosidase from Aspergillus niger, EC

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3.2.1.3, more than 260 U/mL) were purchased from Sigma-Aldrich. The fluorescent

97

dyes including Fluorescein 5-isothiocyanate (FITC), Rhodamine B isothiocyanate

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(RITC), Fluoresceinamine isomer I and Thioflavin T (Th T) were also obtained from

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Sigma-Aldrich. D-Glucose (GOPOD format) assay kit was purchased from Megazyme



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International Ireland Ltd. (Ireland). Polydimethylsiloxane (PDMS, Sylgard184) was

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purchased from Dow Corning Co. (Midland, MI, USA). All other chemicals were of

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analytical or better grade without further purification.

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Preparation of biopolymer stock solutions

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Gelatinized starch paste (GSP, 12.0 wt.%) was prepared by dispersing WCS in

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deionized water (18.2 MΩ cm) and incubating at 95 °C in a water bath for 30 min with

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continuous stirring. GG stock solution (0.8 wt.%) was made by dispersing GG powder

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in deionized water. It was stirred for 12 h at room temperature and then centrifuged at

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4000g for 10 min (25 °C) to remove insoluble materials. Zein was dissolved in ethanol

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solution (80 wt.% ethanol). After stirring for 2 h, it was stored overnight at 4 °C to

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obtain zein stock solution (2.5 wt.% zein). Pectin stock solution (2.4 wt.%) was

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obtained by adding pectin powder in deionized water, and it was stirred for 12 h before

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use. All the solutions were adjusted to pH 7.0 with 1 N NaOH.

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Synthesis and characterization of zein particles

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ZPs were prepared according to the anti-solvent precipitation method described by

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Zhong and Jin with slight modification.31 20 mL of zein stock solution was injected into

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50 mL water with and without pectin using a syringe while stirring at 1000 r/min. After

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15 min, ethanol was removed from the suspension using a rotary evaporator (RV 10

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digital, IKA-Works Inc., Staufen, Germany). The final concentration of zein in the

119

suspensions were 1.0 wt.%, and pectin ranged from 0 to 2.0 wt.%. The ratios between

120

zein and pectin were 1:0, 1:0.1, 1:0.5, 1:1.0, 1:1.5 and 1:2.0, coded as ZP0, ZP1, ZP2,

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ZP3, ZP4 and ZP5, respectively.


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Size distribution, hydrodynamic diameters (Dh) and zeta-potential of the obtained

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particles were determined by dynamic light scattering with a Nano ZS Zetasizer

124

(Malvern Instruments, Worcestershire, U.K.). All the measurements were carried out at

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25 °C using a zein concentration of 2 mg/mL.

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Wettability of ZPs was analyzed by observing their three-phase contact angle (θ)

127

at the WCS/GG interface according to the gel trapping technique (GTT) described by

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Paunov32 with some modifications. Firstly, 10 g of diluted GSP (2 wt.%) was placed in

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an aluminium specimen box (φ = 3 cm), and 5 g of GG solution (0.8 wt.%) was then

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gently dropped on its surface to form a WCS/GG interface. Secondly, 0.6 mL of ZPs

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suspension was carefully injected into the bottom starch phase without disturbing the

132

interface. It was then left at room temperature for 4 h to ensure the adsorption of ZPs at

133

the interface. Afterwards, 1.25 mL of sodium borate solution (0.8 wt.%) was injected

134

into the top GG phase and the mixture was incubated for 12 h, inducing the gelation of

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GG and trapping the particles at the interface. Thirdly, the gelled GG phase was

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removed from the box, and the aqueous starch phase was carefully removed. Then, 1 g

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of PDMS was poured on the top of the surface covered by the particles and cured for 48

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h at room temperature. Finally, the PDMS layer was peeled off and washed with

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deionized water to remove the GG gel residues. The particles trapped on PDMS was

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observed using a FE-SEM (LEO 1530 VP, Carl Zeiss Microscopy GmbH, Jena,

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Germany) at an accelerating voltage of 5.0 kV.

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Preparation and microstructure observation of WCS/GG emulsions

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GSP (0.5-5.0 wt.%), GG (0.3 wt.%), and ZPs suspension (0-1 wt.%) were mixed



144

using a mini-shaker to prepare WCS/GG emulsion. pH was maintained at 7.0. The

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resulting emulsions were imaged using an inverted microscope system (IX 53,

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Olympus, Tokyo, Japan) equipped with a color and monochrome camera (DP 73,

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Olympus, Tokyo, Japan).

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For the WCS/GG mixtures over a range of concentrations in the absence of ZPs, a

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phase diagram was plotted by visualizing macroscopic phase separation of the two

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polysaccharides. To distinguish one biopolymer phase from the other that had similar

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appearance, WCS and GG were covalently labeled by fluorescence markers according

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to the method described by Schmitt and his co-workers.33 25 µL of FITC (2 wt.%) and

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RITC (2 wt.%) dissolved in dimethyl sulfoxide (DMSO) were added to 100 ml GSP

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and GG stock solution, respectively. A gentle stirring (400 r/min) for 1.5 h was then

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conducted to complete the labeling. The mixtures of the two labeled polysaccharides

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were placed in test tubes and centrifuged at 1600g for 1 h (25 °C) to accelerate phase

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separation, which were then macroscopically observed. Furthermore, the emulsion type

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(WCS-in-GG or GG-in-WCS) was determined by observing the microstructure of the

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WCS/GG mixtures. In this case, Fluoresceinamine isomer I was used as the marker for

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WCS. 100 g of GSP (1 wt.%) was sequentially mixed with the dye (3 mg in 3 mL

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DMSO), cyclohexylisocyanide (25 µL) and acetaldehyde (30 µL) under magnetic

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stirring (400 r/min) for 3 h at room temperature. Following removing the excess dye by

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washing with ethanol solution (80 wt.%) and dialysis for 48 h at 4 °C, the labeled GSP

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was lyophilized and stored at -20 °C in darkness before use. The mixture of GG and

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labeled GSP was imaged with the inverted microscope system. The light source was a


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mercury lamp (U-HGLGPS, Olympus, Tokyo, Japan) with an excitation wavelength at

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460-495 nm. The fluorescence intensity was recorded between 510-550 nm.

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For the WCS/GG emulsion stabilized by ZPs, the microstructure was investigated

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with a confocal laser scanning microscope (CLSM, TCS SP5, Leica Microsystems Inc.,

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Heidelberg, Germany). The fluorescence dye Th T (0.01 wt.%), which was able to label

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zein in ZPs, was added to the emulsions prepared according to the procedure described

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above. The light source was an argon laser with excitation wavelength at 458 nm. The

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fluorescence intensity was recorded between 470 and 560 nm. The diameter of the

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droplets in the emulsions were determined from over 8 CLSM images by taking the

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average over calculations more than 100 droplets using the software LAS AF Lite 2.6.3

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(Leica Microsystems CMS GmbH, Germany).

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In vitro digestion analysis

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A two-stage in vitro digestion process that stimulated gastric followed by small

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intestinal environments was conducted using the WCS/GG mixtures according to the

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methods reported by Englyst34 and Zou35 with some modifications. 18 mL of the

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WCS/GG mixture and 5 glass balls in a polypropylene screw-cap tube were

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equilibrated in a shaking water bath at 37 °C for 10 min. After pH was adjusted to 2.0, it

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was then incubated at 37 °C for another 30 min with the addition of 1 mL pepsin

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solution (5 mg/mL, 0.02 N HCl) and shaking at 160 r/min. Afterwards, 5 mL of acetate

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buffer (pH 6) was added to adjust the mixture to pH 6.0, following by addition of 1 mL

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mixed enzyme solution (Pancreatin, 3×103 USP; Amyloglucosidase, 40 U). The

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reaction mixture continued to be incubated at 37 °C with shaking (160 r/min). At



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specific times, 0.5 mL aliquots were removed with a pipette and mixed with 1 mL

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ethanol at room temperature to deactivate the enzymes. This solution was then

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centrifuged at 1500g for 10 min. The glucose content in the supernatant was determined

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using the GOPOD kit.

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The starch digestion results in this work were fitted to a first-order enzyme kinetic equation:

Ct = C∞ ( 1 − e-kt )

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(1)

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Ct is the amount of digested starch at time t (min), which is expressed as the percentage

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of the total starch. C∞ is the corresponding amount at the end point, and k (min-1) is the

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digestibility rate constant. A Logarithm of Slope (LOS) method proposed by

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Butterworth and Ellis36,37 was used to describe the digestibility curves. The LOS plots

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are obtained from equation (2) which is the first derivative of equation (1) in

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logarithmic form. With the linear plot, k and C∞ are calculated from the slope and

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intercept of the curve.

ln(dC/dt) = −kt + ln(C∞ k)

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(2)

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In practice, two stages with rapid and slower digestive rates existed during starch

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hydrolysis in this work. k and C∞ are calculated according to the plots in the

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corresponding linear stage. To identify the two stages, equation (1) is expressed as:

t ≤ tint , Ct = C1∞ ( 1 − e-k t ) t ≥ tint , Ct = Cint + C2∞( 1 − e -k ×(t − t ) ) 1

2

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int

(3)

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where, tint is the intersection time of the two stages, Cint is the amount of digested starch

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at time tint.


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Statistical analysis

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All measurements were conducted at least in triplicate. An analysis of variance

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(ANOVA) of the data was carried out using IBM SPSS Statistics 19.0, and Tukey's

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honestly significant difference test was used for comparison of the means of the three

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replicate values among the treatments using a level of significance of 5%.

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Results

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Phase diagram

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The occurrence of phase separation between the two polysaccharides induced by

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their incompatibility is the prerequisite for the formation of W/W emulsion. To find out

218

suitable combination for W/W emulsion, phase diagram (Figure 1) was investigated

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first. As expected, macroscopic and microscopic phase separation occurred depended

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on the amount and ratio of the two polysaccharides. When the concentrations of WCS

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and GG exceeded 1 wt.% and 0.15 wt.% respectively, macroscopic phase separation

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appeared. In these mixtures, droplets from one polysaccharide-rich phase dispersed in a

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continuous phase that was rich in the other polysaccharide. Obviously, WCS-in-GG

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emulsion is in line with the requirement of this work. To involve more starch in this

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structure, the mixture contained 2 wt.% WCS and 0.3 wt.% GG was used in the further

226

investigation.

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W/W emulsion

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Due to the lack of effective stabilizer, W/W emulsion systems is rarely used.

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With the aid of ZPs, WCS/GG emulsion was obtained in this work. The cycle from

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formation to disintegration of WCS/GG emulsion had been recorded in the form of



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video. It can be found in the Supporting Information (Video S1), and the images that

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exhibit the transformation of the emulsion during some key points are shown in Figure

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2. For the WCS/GG mixture without stabilizers, the emulsion-like pattern appeared 450

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s after the two polysaccharides were mixed and placed on the microscope. This

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structure did not last long. No droplets can be found in the image recorded at 1200 s,

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which is the result of macroscopic phase separation at this time. Apparently, this time is

237

too short for the mixture traveling through the gastrointestinal tract within hours during

238

digestion. The stability of this emulsion can be greatly improved by the addition of ZP3

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(0.2 wt.%), which is confirmed by the Video S2 (Supporting Information) and the

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corresponding images in Figure 2. In this case, the presence of the particles delayed the

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formation of W/W emulsion. The droplets could not be seen until 2700 s. However,

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they had not disappeared even after the observation was finished at 9000 s. With ZPs,

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the time that the emulsion droplets remained stable was substantially extended.

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Effects of ZPs on the stability of WCS/GG emulsion

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Prior to subjecting WCS/GG emulsion to the analysis of starch hydrolysis kinetics,

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the effects of ZPs on the stability and microstructure of W/W emulsion should be

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clarified. First, emulsions with a range concentration of ZP3 (0-0.3 wt.%) were

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prepared. They were kept at room temperature under observation for one week. Photos

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taken during this process were shown in Figure 3. No macroscopic phase separation

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occurred in all the freshly prepared emulsions. The increased turbidity of the emulsions

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was due to the addition of ZPs. After left standing for 1 day, their appearance began to

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change. They were no longer composed of single phase. Creaming and macroscopic


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phase separation happened in these emulsions, leading to the formation of a GG-rich

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phase at the top and a turbid emulsion layer at the bottom. This evolution became more

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pronounced as the extension of storage time. As more ZPs was added, the mixture had

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greater volume of emulsion layer. It suggested that macroscopic phase separation in the

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mixtures could be slowed when more ZP3 were used in the emulsions.

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Meanwhile, microstructure of the freshly prepared emulsions was observed via

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CLSM. The obtained images were illustrated in Figure 4. The WCS-rich droplets were

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easy to identify even in the bright-field images. Thus, their size could be calculated

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using the software LAS AF Lite (version 2.6.3). The average diameter of the droplets

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decreased from 179 ± 51 µm to 90 ± 20 µm as the addition of ZP3 increased from

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0.0125 wt.% to 0.30 wt.%. Smaller droplets, which leads to larger surface area of the

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interface, are favourable for the improvement of emulsion stability. The self-assembly

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of zein during the preparation of ZPs has been reported to be the result of β-sheet

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transformation, orientation, alignment, and packing.38 Th T, which could bind closely

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to β-sheet-rich structures in protein, was used to label ZPs and visualize their location in

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the emulsions. The resultant CLSM images of this fluorescent dye signal in Figure 4

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revealed that most ZP3 appeared around the droplets rather than in the external phase.

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And more particles accumulated at the WCS/GG interface in the emulsion with more

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ZP3. In this case, more barriers were provided to the droplets, leading to the formation

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of smaller droplets and brought better stability for the emulsion.

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To modify the functionalities of ZPs, positively charged zein was mixed with

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various ratios of pectin (zein/pectin, from 1:0 to 1:2) a negatively charged



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polysaccharide during anti-solvent precipitation. The resulting complex particles

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possessed different size and wettability. First, ZPs became larger as more pectin was

277

involved during preparation (Figure 5a). The particle size of those without pectin (ZP0)

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was 124.1 ± 0.1 nm. And it gradually increased to 200.4 ± 0.2 nm as the zein/pectin

279

ratio increased to 1:0.5 (ZP2). Once the ratio exceeded this value, a sudden increase of

280

the particle size was observed. For ZP3 prepared by the ratio of 1:1.0, it was 695.6 ± 0.3

281

nm. Further increase of pectin (zein/pectin, 1:1.5 and 1:2.0) made the particles become

282

so large that their size was out of the measurement range. As the amount of pectin was

283

in excess of zein, the properties of the resultant particles were dominated by the

284

polysaccharide, resulting in the growth of complex particles.

285

The behavior of ZPs at WCS/GG interface also followed this trend. The particle

286

wettability was analyzed by observing their surface structuring adsorbed at WCS/GG

287

interface. As shown in the SEM images (Figure 5b), particles were jammed in between

288

the two phases. The bottom one is the PDMS layer. The part of ZPs immersed in this

289

layer was in contact with WCS in the original system. And the visible part was in

290

contact with GG. Although accurate three-phase contact angles could not be calculated

291

from these images, the variation trend of ZPs wettability was easy to summarize. Most

292

part of ZP0 immersed in the PDMS layer. More visible part emerged in the ZPs

293

synthesized with more pectin. It suggested that the addition of pectin obviously

294

changed the particles wetting properties in the two polysaccharide phases., turning the

295

WCS compatible particles into those had a strong affinity with GG.

296

This change of particle wettability certainly affected the location of ZPs in the


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WCS/GG emulsion, which was reflected in the CLSM images (Figure 6a). In the

298

emulsion stabilized by ZP0 which had better affinity with WCS and poorly wetted in

299

GG phase (Figure 5b), most particles appeared inside the WCS-rich droplets and

300

accumulated at the interface. Few were found in the GG phase. For ZP3, their

301

wettability in GG phase was greatly improved (Figure 5b). By comparison, their

302

affinity towards the two polysaccharide liquids was more similar. Therefore, ZP3 were

303

excluded from the two phases and adsorbed at the interface, resulting in a high surface

304

coverage for the droplets (Figure 6a). When the droplets touched each other, ZP3 at the

305

W/W interface tended to enter the external continuous phase rather than the interior

306

droplet phase. They could inhibit the coalescence of the droplets and became an

307

effective barrier for the WCS-rich droplets. As shown in Figure 6b the observation on

308

emulsion stability, the volume of the emulsion layer in the samples declined during

309

storage due to the separation of the two phases. In contrast, this volume of the sample

310

stabilized by the ZPs prepared with more pectin was greater. This implied that the

311

presence of pectin in ZPs helped to improve the storage stability of the resulting

312

WCS/GG emulsion. However, it had not been further improved in the cases of ZP4 and

313

ZP5 when compared with that of ZP3. This might be attributed to the wettability and

314

interfacial adsorption of the particles. As can be seen in the CLSM image of the sample

315

with ZP3 (the inset of Figure 6a), an uninterrupted colored loop appears around the

316

WCS-rich droplet, indicating that the surface of the droplet was full of ZP3. In the case

317

of ZP5, the colored loop around the droplet is no longer intact. Many gaps can be found,

318

suggesting the decline of the particle interfacial adsorption. For ZP5, strong affinity



319

with GG phase, poor wettability in WCS phase and enlarged particle size were

320

responsible for the reduced adsorption energy. And particle desorption from the

321

interface became more easily. Therefore, modification of zein particles with

322

appropriate amount of pectin is necessary. Considering the interfacial coverage and

323

wetting properties, ZP3 was used to prepare WCS/GG emulsion in the following

324

section.

325

Starch digestion

326

Four cases including WCS, mixture of WCS and GG, WCS/GG emulsion with

327

ZP0 and WCS/GG emulsion with ZP3, which were the representative of various

328

compositions and preparation procedures, had been subjected to the investigation of

329

starch hydrolysis kinetics in vitro, so as to clarify the effects of the formed structure.

330

Their starch digestion curves were shown in Figure 7. After fitting to a first-order

331

equation, the Logarithm of Slope (LOS) plots indicated the existence of two distinct

332

linear phases, in which starch was digested at a rapid and a slow rate. The rate constant

333

in the former phase was nearly five times of that in the latter (Table 1). Certainly, starch

334

in the sample contained only WCS (2.0 wt.%) was digested in a faster rate and a higher

335

degree, when compared to the other three samples. Eventually, about 87.9% starch was

336

hydrolyzed during the test. The WCS/GG (2.0 wt.%/0.3 wt.%) mixtures with and

337

without ZP0 (0.2 wt.%) had similar curves. By comparison, their rate constants in rapid

338

and slow phase decreased by about 60% and 50%, respectively. In the case of WCS/GG

339

mixture with ZP3, the rapid phase was negligible. About 74.7% of the starch inside was

340

digested at a rate similar to those in the slow phase of the above cases. Evidently,


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341

addition of other substances like GG and ZP0, as well as W/W emulsion structuring

342

could slow down the release of glucose from gelatinized WCS. These additions

343

increased the viscosity of the systems in contrast to that only contained WCS (Figure

344

S1), which might delay starch digestion to some extent. However, the starch digestion

345

kinetics of the WCS/GG emulsion stabilized by ZP3 is significantly distinct from

346

those of the WCS/GG mixtures with and without ZP0. Since these three samples had

347

similar viscosity ((Figure S1), it indicated that the formation of W/W structure played

348

a more critical role in the inhibitory action of glucose release. This structure set up

349

barriers for the interaction between the enzymes participated in the starch hydrolysis

350

and WCS the substrate. And this effect depended on the protection for WCS provided

351

by the particles. The longer time this structure was maintain, the lower digestion rate

352

and degree were obtained. The difference between WCS/GG mixtures stabilized by

353

ZP0 and ZP3 proved this point. Through CLSM observations (inlet of Figure 7), it was

354

noted that most of the former particles dispersed inside the WCS-rich droplets after

355

simulative gastric digestion. For the latter particles, colored loops were founded around

356

the droplet surface, suggesting that ZP3 still located at W/W interface and provided

357

effective protection for the WCS-rich droplets in the following simulative intestinal

358

digestion. As this structure gradually collapsed, glucose was released at a slow rate.

359

This also revealed that the modification of zein particles with pectin in the fabrication

360

of WCS/GG emulsion structure was of importance.

361

In conclusion, a strategy for reducing the rate of glucose release from starch via

362

fabricating colloidal structure was proposed in this work. Due to the incompatibility of



363

two biopolymers, microscopic phase separation appeared in the mixture of WCS and

364

GG. The resulting WCS-in-GG emulsion was stabilized by ZPs which was obtained by

365

anti-solvent precipitation. Particle wetting properties could be changed by the addition

366

of pectin during particle preparation. As the ZPs had similar wettability in the two

367

polysaccharide liquids, their adsorption at the W/W interface and the coverage on the

368

WCS-rich droplets both increased. By means of this W/W emulsion system, WCS was

369

wrapped around by particles which acted as the barriers for its contact with the enzymes

370

during amylolysis. Eventually, this structure changed the enzyme kinetics in starch

371

digestion, and a sustained release of glucose was observed.

372

Abbreviations used

373

WCS, wax corn starch; GG, guar gum; GSP, gelatinized starch paste; GI, glycemic

374

index; ZPs, zein particles; ZP0, ZP1, ZP2, ZP3, ZP4 and ZP5, the ZPs prepared with

375

zein/pectin ratio of 1:0, 1:0.1, 1:0.5, 1:1.0, 1:1.5 and 1:2.0, respectively; W/W,

376

water-in-water; O/W, oil-in-water; DLS, dynamic light scattering; GTT, gel trapping

377

technique; CLSM, confocal laser scanning microscope; SEM, scanning electron

378

microscope; LOS, Logarithm of Slope; FITC, fluorescein 5-isothiocyanate; RITC,

379

rhodamine B isothiocyanate, Th T, Thioflavin T; PDMS, polydimethylsiloxane

380

Acknowledgment

381

This work was supported by the National Natural Science Fund of China (no.

382

31501425 and 31771923).

383

Supporting Information description

384

Video of formation and decomposition of WCS/GG (2 wt.%/0.3 wt.%)


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385 386

emulsions with and without ZP3 (0.2 wt.%) Viscosity of WCS (2 wt.%), WCS/GG mixture (2 wt.%/0.3 wt.%), WCS/GG

387

mixtures (2 wt.%/0.3 wt.%) with ZP0 (0.2 wt.%) and ZP3

388

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Figure captions Figure 1 Phase diagram of WCS/GG mixture. The grey circles indicate the mixtures with single phase. The green circles correspond to the WCS-in-GG emulsions, and the reds correspond to the GG-in-WCS emulsions. In the emulsions, the top GG phase is red colored by RITC, and the bottom WCS phase is green colored by FITC. In the microscopic images, the colored area derived from the fluorescent signal provide by Fluoresceinamine, isomer I which had been labeled with WCS. Figure 2 Optical microscopy images of WCS (2.0 wt.%) and GG (0.3 wt.%) mixtures with and without ZP3 (0.2 wt.%) at different times after they were mixed and placed on the microscope. Figure 3 WCS/GG (2.0 wt.%/0.3 wt.%) emulsions stabilized by various concentrations of ZP3 (from left to right: 0, 0.0125, 0.025, 0.05, 0.10, 0.20, 0.30 wt.%) as a function of time. Figure 4 CLSM images of ZPs signal (left) and bright-field (right) of WCS/GG (2.0 wt.%/0.3 wt.%) emulsions stabilized by various concentrations of ZP3. The average diameters of the WCS-rich droplets and their standard deviation (see insets) were calculated from more than 100 droplets in 8 images using the software LAS AF Lite (version 2.6.3). The scale bars indicate 250 µm. Figure 5 (a) Size distributions of ZPs prepared with different zein/pectin ratios. (b) SEM images of ZPs trapped on PDMS from the WCS/GG interface with the gel trapping technique. The visible part of ZPs has been in contact with the GG phase in the original system.



Figure 6 CLSM images (a) and storage stability (b) of the WCS/GG (2.0 wt.%/0.3 wt.%) emulsions stabilized by ZPs (0.2 wt.%) prepared with various zein/pectin ratios (panel b, from left to right: 1:0, 1:0.1, 1:0.5, 1:1.0, 1:1.5, 1:2.0). The average diameters of the WCS-rich droplets and their standard deviation (see insets) were calculated from more than 100 droplets in 8 images using the software LAS AF Lite (version 2.6.3). The scale bars in panel (a) indicate 250 µm. Figure 7 Starch digestion curves for WCS, WCS/GG, WCS/GG with ZP0 and WCS/GG with ZP3 that had been hydrolyzed by pepsin. The concentration of WCS and GG in all samples was 2.0 wt.% and 0.3 wt.%, and the amount of ZPs was 0.2 wt.%. Inlet: CLSM images of WCS/GG emulsions stabilized by ZPs before (0 min) and after (240 min) the starch digestion. The scale bars indicate 250 µm.


Page 26 of 35

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Tables Table 1 Values of variables calculated using the LOS method for WCS, WCS/GG, WCS/GG with ZP0 and ZP3 a

C1∞ (%)

k1 (min-1)

Single or slow phase k2 C2∞ (%) (min-1)

WCS

68.6

0.145

19.3

0.022

20

65.3

87.9

WCS/GG

54.0

0.058

25.4

0.011

30

58.3

79.4

WCS/GG, ZP0

59.9

0.057

30.8

0.012

30

50.9

90.7

WCS/GG, ZP3

-b

-b

74.7

0.019

-b

-b

74.7

Rapid phase

Intersection

Total

Tint (min)

Cint (%)

C∞ c (%)

a

Values are calculated from LOS plots with one or two-phases.

b

Indicates that starch amylolysis only occurred in a single-phase.

c

Total C∞ is the sum of C1∞ and C2∞, representing the total degree of starch digestion



Figure graphics Figure 1


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Figure 2



Figure 3


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Figure 4



Figure 5 (a)

(b)


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Figure 6 (a)

(b)



Figure 7


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Graphic for table of contents


Slowing the Starch Digestion by Structural Modification through

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