In Vitro Digestibility of Nanoporous Wheat Starch Aerogels - Journal of

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In Vitro Digestibility of Nanoporous Wheat Starch Aerogels Ali Ubeyitogullari, Sandrayee Brahma, Devin Jerrold Rose, and Ozan Ciftci J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b03231 • Publication Date (Web): 21 Aug 2018 Downloaded from http://pubs.acs.org on August 22, 2018

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Journal of Agricultural and Food Chemistry

In Vitro Digestibility of Nanoporous Wheat Starch Aerogels

Ali Ubeyitogullari, Sandrayee Brahma, Devin J. Rose, and Ozan N. Ciftci* Department of Food Science and Technology, University of Nebraska-Lincoln Lincoln, NE 68588-6205, USA

*Corresponding author * Ozan N. Ciftci E-mail: [email protected] Tel: +1-402-4725686

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ABSTRACT

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This study reports the in vitro digestibility of starch aerogels for the first time. The relative

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crystallinities of the wheat starch aerogels (WSAs) produced at gelatinization temperature of 120

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°C (WSA-120C), 130 °C (WSA-130C) and 120 °C with addition of STMP (WSA-STMP), and

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xerogel were similar. However, WSA-120C showed the highest amylose-lipid complex content.

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The addition of STMP created some cross-linked starch with phosphorus content of 0.023%.

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Resistant starch (RS) contents of the WSA-STMP (33.5%) and xerogel (26.9%) were higher than

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the other samples when they were uncooked prior to digestion. Nevertheless, the RS contents of

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the WSA-STMP and xerogel decreased drastically with cooking. RS contents of WSA-120C and

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WSA-130C were stable with cooking and provided 4.5 and 3.0-fold increase in the RS content,

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respectively. WSA is a promising functional food ingredient with high RS content even after

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cooking.

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KEYWORDS: Wheat starch, aerogel, digestibility, resistant starch, supercritical, nanoporous.

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INTRODUCTION

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Organic aerogels have been attracting interest for various food, nutraceutical, and pharmaceutical

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applications due to their high surface area, lightweight and porous structure. Recent studies have

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focused on the application of aerogels to enhance the solubility and/or bioavailability of poorly

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water-soluble bioactive compounds by impregnation into the aerogel matrix.1-2 To illustrate,

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impregnation of vitamin D3 into an alginate aerogel resulted in 20X higher solubility compared

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to the crude vitamin D3 due to the decrease in the crystallinity caused by impregnation into the

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aerogel.3 Furthermore, inorganic silica aerogels have been extensively studied to improve and

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control the release of drugs like ketoprofen, griseofulvin, benzoic acid and paracetamol.1, 4

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Among polysaccharide-based aerogels, starch aerogels have received much attention due to their

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low cost, renewability, stability upon storage, biodegradability, and biocompatibility. Recently,

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García-González et al.5 investigated supercritical carbon dioxide (SC-CO2)-aided impregnation

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of ketoprofen and benzoic acid into corn starch aerogels. Impregnated drugs had a less

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crystalline structure compared to their free forms and impregnation into aerogels provided a

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controlled release in both simulated gastric and intestinal fluids.5

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Wheat is a great source of starch to produce aerogels with extraordinary properties for

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food applications.6 In our previous studies, wheat starch aerogels were used to generate low-

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crystallinity phytosterol nanoparticles7-8 where the solubility of the phytosterols was improved

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37-fold compared to crude phytosterols by impregnation into the nanoporous wheat starch

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aerogels (WSAs).7 Although various aerogels have been produced with the aim of improving the

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bioavailability of lipophilic bioactives, none of those aerogels have been investigated for their

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digestibility which is critical not only for the release of the bioactives but also for the nutritional

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value of the aerogel matrix.



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In recent years, there have been considerable research efforts to increase the RS content

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of starches9-13 because of the potential of RS to improve human health by protecting against

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diseases like colon cancer, type 2 diabetes, and obesity.14-16 The rate and extent of starch

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digestion are affected by several factors such as fine structure of amylopectin, surface

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morphology, porosity, amylose-lipid complexes and crystallinity.17 The physical properties that

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affect starch digestion are likely influenced by starch aerogel production steps, namely,

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gelatinization, solvent exchange, retrogradation and drying.6 Moreover, chemical modification of

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starch with sodium metaphosphate (STMP) affects not only the digestibility but also the physical

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properties.18 Our hypothesis was that addition of STMP into the aerogels would affect the

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structure and also digestibility of the aerogels.

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Therefore, the main objective of this study was to investigate the in vitro digestibility of

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WSA. Specific objectives were to: (a) produce and characterize WSA by both gelatinization

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upon heating and using STMP as a chemical cross-linker; and (b) investigate the effect of surface

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area and chemical cross-linker on the digestibility of WSA. Wheat starch and wheat starch

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xerogels (air-dried) were also evaluated for their digestibility and compared with those of WSAs.

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MATERIALS AND METHODS

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Materials. Wheat starch was kindly donated by Manildra Milling Corporation (Hamburg,

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IA, USA). Wheat starch was composed of 25% amylose and 75% amylopectin as declared by the

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supplier. Food grade STMP was obtained from Innophos (Cranbury, NJ, USA). Ethanol (100%)

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was purchased from Decon Laboratories, Inc. (King of Prussia, PA, USA). CO2 (99.99% purity)

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was supplied by Matheson Tri-Gas, Inc. (Lincoln, NE, USA). KH2PO4 (purity ≥ 99%) was

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purchased from MP Biomedicals, LLC (Solon, OH, USA). Thermostable α-amylase (from

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Bacillus licheniformis, 3000 U/mL), amyloglucosidase (from Aspergillus niger, 3260 U/mL) and 4 ACS Paragon Plus Environment

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glucose oxidase-peroxidase assay kit were purchased from Megazyme (Bray, Ireland). Pepsin

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(from porcine gastric mucosa, ≥250 units/mg solid) and pancreatin (from porcine pancreas,

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neutral protease: 208 USP units/mg solid; α-Amylase: 223 units/mg solid; lipase: 38.5 USP

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units/mg solid) were obtained from Sigma-Aldrich (St. Louis, MO, USA).

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Formation of nanoporous starch aerogels. Aerogels were produced according to the

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method of Ubeyitogullari and Ciftci6 where the processing parameters were optimized for the

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highest surface area. Aerogel formation consisted of three main steps, namely, gelatinization,

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solvent exchange, and SC-CO2 drying. Briefly, starch dispersions (10 wt.%) were gelatinized in

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a closed high-pressure reactor (4520 Bench Top Reactor, Parr Instrument Company, Moline, IL,

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USA) at 120 °C with a mixing rate of 600 rpm for 20 min to form a hydrogel. The hydrogels

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were stored at 4 °C for 48 h, and then the water in the hydrogel was replaced with ethanol using a

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solvent exchange step to obtain an alcogel. Solvent exchange took place by soaking the

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hydrogels into 30, 50, 70, and 100% (v/v) ethanol solutions for 1 h and then keeping in 100%

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ethanol for 24 h. Finally, the alcogels were dried in a custom-made laboratory scale SC-CO2

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drying system at 40 °C and 10 MPa for 4 h to obtain the aerogels (WSA-120C). Details of the

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SC-CO2 drying system and its operation were reported previously.8

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In addition to WSA-120C, the wheat starch dispersion (10 wt.%) was also gelatinized at

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130 °C and converted into an aerogel (WSA-130C) as described above to study the effect of

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surface area on digestibility. STMP-crosslinked aerogels (WSA-STMP) were formed by

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incorporating 1 wt.% (starch basis) STMP into wheat starch dispersion (10 wt.%), followed by

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SC-CO2 drying to form aerogels. Xerogels were generated by air drying of the alcogels at room

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temperature (21 °C) until constant weight was achieved.



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The samples were stored at room temperature (21 °C) until characterized. The surface

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area, pore size, pore volume, density, and porosity of the samples were determined according the

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methods described previously.6 Briefly, Brunauer–Emmett–Teller (BET) surface area and

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Barrett-Joyner-Halenda (BJH) pore size and pore volume were determined from nitrogen

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adsorption-desorption isotherms measured at -196 °C on a surface area and porosimetry system

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(ASAP 2020, Micromeritics Instrument Corporation, GA, USA). Samples (0.06-0.3 g) were

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degassed at 115 °C for 4 h under vacuum prior to analysis. Surface area was calculated using

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multipoint BET adsorption characteristics at a relative pressure between 0.05 and 0.3, whereas

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pore size and volume were determined at a relative pressure of >0.35. Moreover, density of the

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samples was determined by measuring the dimensions of the monoliths and weighing them. The

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porosity was calculated using the bulk density ( ) of the samples and the true density ( =

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. )

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of the starch using Eq 1.7

% = 1 − ∗ 100

(1)

Morphology. The morphology of the samples (WSA-120C, WSA-130C, WSA-STMP

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and xerogel) was examined using a field emission scanning electron microscope (S4700 FE-

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SEM, Hitachi, Tokyo, Japan).7 The SEM was operated in low vacuum mode at 5 kV and 15 mA.

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The specimens were prepared by cutting 1-mm thick cross-sections from the center of the

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monoliths using a double-edged razor blade. Then, the specimens were placed on double-side

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conductive carbon tape mounted onto circular aluminum SEM stubs. The samples were sputter-

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coated with a chromium layer under vacuum (Desk V HP TSC, Denton Vacuum LLC, NJ, USA)

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for 5 min prior to analysis. The thickness of the fibrils was calculated using ImageJ v. 1.50i


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software (public domain, National Institutes of Health, Bethesda, MD, USA) from the captured

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SEM images.

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Crystallinity. X-ray diffraction (XRD) analysis was performed to study the crystallinity

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of the samples.7 XRD patterns of the samples were measured using a PANalytical Empyrean

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Diffractometer (Empyrean, PANalytical B.V., Almelo, Netherlands). The instrument was

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equipped with PIXcel3D detector and operated with 1D detection at 45 kV and 40 mA. The

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samples were ground and sieved through mesh # 20 (0.85 mm screen) prior to analysis. The

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powdered samples were placed on the sample holder and continuously scanned between 2° and

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40° (2θ) at a scan speed of 0.927 °/min with a step size of 0.05°. The sample holder was spun at

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a rate of 3.75 rpm throughout the analysis. Degree of crystallinity was calculated from the ratio

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of the area of the crystalline part to the total area of amorphous and crystalline parts using

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Software OriginPro 2017 (OriginLab Corporation, Northampton, MA, USA).6

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Fourier-transform infrared spectroscopy. Attenuated total reflectance Fourier-

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transform infrared (ATR-FTIR) spectra of the samples was determined using a Thermo Scientific

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Nicolet 380 spectrometer (Thermo Scientific, Waltham, MA, USA). The samples were scanned

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between 4000 and 400 cm-1. Each spectrum was collected by averaging 128 scans at a spectral

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resolution of 4 cm-1. Nicolet Omnic 8.3 software was used for deconvolution from 1200 to 800

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cm-1 with a half-bandwidth of 19 cm-1 and an enhancement factor of 1.9.19 The absorbance ratios

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at 1047/1022 cm-1 and 1022/995 were obtained from the deconvoluted spectra to estimate the

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short-range ordered structure of the samples.

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Sample preparation for NMR analysis. WSA-STMP was digested prior to the 31P

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NMR analysis using the method of Sang et al.11 Briefly, WSA-STMP was ground and sieved

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through a 0.85 mm screen (mesh # 20). One gram of powdered WSA-STMP was digested with 7 ACS Paragon Plus Environment


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100 µL of heat-stable α-amylase in 50 mL of 2.0 mM calcium chloride. Digestion took place in a

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boiling water bath for 30 min. Then, another 100 µL of heat-stable α-amylase was added and the

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digestion was repeated. After α-amylase digestion, the mixture was cooled, and the pH was

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adjusted to 4.5. Then, the mixture was further digested with 200 µL of amyloglucosidase at 60

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°C for 1 h. The mixture was adjusted to pH 7.0 and centrifuged at 4000 g for 10 min. Finally, the

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supernatant was freeze-dried for 24 h using a Freezone 6 freeze dryer (Labconco, Kansas City,

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MO, USA). 31

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P NMR spectra of the digests of WSA-STMP. The freeze-dried digest of WSA-

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STMP (100 mg) was dissolved in 1 mL of deuterium oxide containing 20 mM EDTA and

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0.002% sodium azide, and the solution was adjusted to pH 8.0 with 0.1 M NaOH. KH2PO4 was

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added as internal standard into the solution. The proton-decoupled 31P NMR spectra were

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acquired on a Bruker Avance III-HD 700 MHz spectrometer equipped with a quadruple

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resonance QCI-P cryoprobe (1H, 13C, 15N, 31P). The system was operated at 700.17 MHz for 1H

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and 283.43 MHz for 31P, respectively. The 31P NMR spectra were collected with 128 scans using

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a recycle delay of 2.0 sec and spectral width of 11.4 kHz. The spectra were recorded and

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analyzed with TopSpin 3.5 software.

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Viscosity. The viscosities of the starch slurries were determined using a controlled-stress

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rheometer (Physica MCR 301, Anton Paar GmbH, Graz, Austria). The samples were prepared at

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the same concentration (0.25g/10 mL) level used for the in vitro digestion. Parallel plates with a

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diameter of 50 mm (PP50) were used. The gap size was set to 1.00 mm. Five minutes after the

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sample was loaded, time sweep of 30 min at 37 °C was performed at constant shear rate of 200 s-

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. Duplicate measurements were carried out for each sample.


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In vitro digestion. WSAs and xerogel were ground and sieved through mesh # 50 (0.3

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mm screen), and then subjected to in vitro digestion according to Mkandawire et al.21 In short,

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250 mg of starch sample was dispersed in 2 mL of water in a 15 mL test tube. Two treatments

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were applied before digestion, namely cooked and uncooked. For the former treatment, the test

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tubes containing the samples were cooked in boiling water bath for 20 min with vortex mixing

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several times during the first 5 min of cooking, and then cooled by placing the tubes in a water

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bath at 37 °C. For the latter treatment, the WSAs and the xerogel were simply placed in the

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water bath at 37 °C. To achieve the gastric phase, 4 mL of 3.6% w/v pepsin in 50 mM

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hydrochloric acid was added to each tube, vortex mixed, and incubated for 30 min at 37 °C. Six

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glass beads (6 mm diameter) were subsequently added to the tubes followed by 2 mL of sodium

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acetate buffer (0.5 M, pH 5.2, containing 5 mM calcium chloride). The small intestinal phase

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was initiated by adding 2.05 mL of an enzyme mixture. The enzyme mixture was prepared by

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adding pancreatin in water (15% w/v) on a magnetic stirrer for 10 min and then centrifuged for

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10 min at 4000g. Twenty µL of amyloglucosidase was added per mL of recovered pancreatin

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supernatant. Starch was digested over 2 h at 37 °C with horizontal shaking at 200 rpm. Fifty µL

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of aliquots of slurry were removed from the tube after exactly 20 min and 120 min of digestion

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and mixed with 0.95 mL of absolute ethanol to stop the enzymatic reaction. The mixtures were

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then centrifuged at 5000g for 5 min. Glucose was quantified in the supernatant using the glucose

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oxidase-peroxidase method and was converted to starch content by multiplying by a factor of

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0.9. The starch that was converted to glucose within 20 min of digestion was rapidly digestible

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starch (RDS), after 120 min of digestion was total digestible starch (TDS) and slowly digestible

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starch (SDS) was the fraction of starch digested between 20 and 120 min of in vitro digestion.

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RS was the fraction that remained undigested after 120 min under the experimental conditions



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and was obtained from the difference between total starch (TS) and TDS. A control digestion

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with cooked wheat starch was also performed.

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Statistical analysis. The results were expressed as the mean ± standard deviation.

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Statistical analysis of the data was carried out using Minitab® 16.1.1 software (Minitab Inc.,

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State Collage, PA, USA). Statistical differences among the treatments were determined by

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analysis of variance (ANOVA) and Tukey’s multiple comparison test at a significance level of

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p

In Vitro Digestibility of Nanoporous Wheat Starch Aerogels - Journal of

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