Progress 04/01/23 to 03/31/24
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals? Comparison of the encapsulation efficiency, stability of the encapsulated bioactive compounds, and in vitro release profiles between encapsulated WMS and SLG delivery systems. Evaluate gut microbiota-modulating effects of quercetin and ginger extract-loaded starch micro/nanoparticles.
Impacts
What was accomplished under these goals?
1. Development of a procedure for encapsulating starch granules Firstly, native WMS was modified by using phosphate salts to make its surface negatively charged. Secondly, modified starch was further encapsulated with positively charged zein through electrostatic interaction. The negativelycharged alginatewas then layered as the secondary coating agent. 2. Themorphologicaland structural properties ofnative and encapsulated WMS The morphology of native and encapsulated WMS samples was examined by confocal laser scanning microscope (CLSM 710, Zeiss). 3. In vitro starch digestion kinetics Starch digestion kinetics was measured using a literature method with some modifications. Aliquots of the enzyme solution (porcine pancreatic alpha-amylase and amyloglucosidase) and guar gum solution were added to the gelatinized starch samples, followed by incubation in a water bath (37 °C) with agitation. Aliquots (100 µl) were taken at designated time intervals and mixed with 800 µl of 80% ethanol. The glucose contents in the mixture were determined using a Glucose (GO) assay kit. 4. Preparation of short linear glucan (SLG) WMS was dispersed in an acetic acid buffer solution (0.01mol/L, pH 5.5). The starch slurry was cooked and then completely gelatinized. The temperature was adjusted to 50°C and pullulanase (100U/g dry starch) was added. The mixture was incubated at 50 °C for 12 h. The mixture was centrifuged and the SLG in the supernatant was precipitated using alcohol at a water/ethanol volume ratio of 1:3. 5. Differential scanning calorimetry (DSC) analysis A DSC analyzer (Model 60A, Shimadzu Corporation, Kyoto, Japan) was employed to analyze the thermal behavior of native WMS and SLG. Four milligrams (dry weight basis) of the sample were weighed accurately into an aluminum pan and mixed with DI water (1:1, w/w). The pan was hermetically sealed and equilibrated at room temperature for 24h. Samples were heated from 25 to 130°C at 10°C/min. An empty sealed aluminum pan was used as a reference. The transition temperatures, i.e., onset (To), peak (Tp), conclusion (Tc), andenthalpy of gelatinization (ΔH) were analyzed and calculated. 6. Fourier transform infrared (FT-IR) analysis An FT-IR spectrophotometer (Varian 640, Agilent Technologies Inc., Santa Clara, CA, USA) was employed to measure the spectra of starch samples at the range from 4000 to 400cm−1using air as the background. All spectra were baseline-corrected. Furthermore, the spectra between 1200 and 800 cm−1were deconvoluted. The intensity ratio of 1047 to 1022cm−1(R1047/1022) was used to evaluate the short-range ordered structure of starch samples. Results: Themorphologicaland structural properties ofnative and encapsulated WMS The morphology of native and encapsulated WMS granules was first examined under transmitted light. And then the cross-sectionalimage of the same starch sample was examined by CLSM, with the outer encapsulation layer being covalently labeled with fluorescein-5-isothiocyanate (FITC). The FITC-labeled encapsulation layer appeared green under CLSM. From the mergedimage, it can be clearly seen that the starch granules were fully covered by the encapsulation materials. Kineticanalysisof the digestionprofiles ofnative and encapsulated WMS Comparisonof the percentageof totalglucosereleasedfrom native and zein/alginate encapsulated WMS are depicted inTable 1. The native WMS was hydrolyzed quickly and the release of glucose was fast during the early stage, reachingaplateau within 60 min. On the contrary, the hydrolysis of encapsulated starch was much slower, especially during the first 60 minutes and a sustained glucose release profile was observed. The hydrophobic zein in the encapsulation layer not only reduced the swelling and rupture of starch granules during gelatinization, but also restrict the access of amylolytic enzymes to starch. As zein in the coating layer was gradually hydrolyzed by pancreatin, encapsulated starch granules were gradually exposed and digested. Table 1. Profiles of glucose release percentage for native and zein/alginate encapsulated WMS Incubation time (min) Glucose release percentage (%) 3 min 6 min 10 min 15 min 20 min30 min 40 min 50 min 60 min120 min180 min Native WMS 50 %50% 60% 65% 74% 81% 90% 92% 94% 97% 98% encapsulated starch 44% 53% 56% 61% 66% 72% 74% 78% 77% 78% 82% Thermal transition properties The thermal parameters (To,Tp,Tc, ΔH) of native starch and SLG samples are summarized in Table 2. Native WCS showed a single endothermic peak around 71.2 °C, in agreement with previous studies. Debranched starch presented a significant increase in the transition temperatures (To, Tp, and Tc) as compared to native starch. This is due to the fact that new and more stable crystallites were formed by the SLG within a well-organized structure. Table 2.Gelatinization characteristics of waxy corn starch (WCS) and shortlinearglucan (SLG). To,TpandTcwere onset, peak and conclusion temperature, respectively;Hwas enthalpy of gelatinization Samples To(°C) Tp(°C) Tc(°C) ΔT (°C)ΔH(J/g) WCS 64.2 71.2 75.9 11.7 17.7 SLG 84.6 87.7 94.6 3.4 13.9 The ΔH is a measure of the amount of energy required to dissociate the double helices during gelatinization. As shown in Table 2, SLG had slightly lower ΔH value than that of the control starch. This suggests that debranching modifications could reinforce the structure of the double helices or ordered/crystalline regions. Therefore, less energy was required to dissociate/melt these stable double helices. FTIR Characterization of native WCS and SLG FTIR spectroscopy was used to study the short-range ordered structure of native WCS and SLG. The FT-IR diffractograms revealed that both samples possessed an extremely broad absorption band from 3100 to 3700cm−1, which was attributed to the complex vibrational stretches associated with free, inter- and intra-molecular O-H groups among the neighboring molecules of starch granules. The peak at 2930cm−1wasrelatedtothe asymmetric stretching vibration of C-H bond of the glucose unit. The peak at 1640cm−1was due to tightly bound water that was predominantly present in the noncrystalline region. In addition, the FT-IR spectra also showed three characteristic bands in the fingerprint region (1000-1200cm−1), which correspond to the stretching vibration of C-O. There was no apparent new absorption peak in the FT-IR spectra of debranched starch but the intensity of certain absorption peak was different, indicating no new chemical bonds formed from the debranching process. However, the peak intensity of -OH groups in SLG was evidently weakened, possibly due to a reduction in free -OH groups caused by the increase in hydrogen bonds between SLG molecular chains. Intensityof characteristicpeakat 2930cm−1 was also reduced by debranching but the intensities of the peak between 1200 and 1400 cm−1was enhanced, indicating that the α-1,6-glycosidic bonds were hydrolyzed by pullulanase, which could increase the quantities of CH2OH. The peaks at 1047cm−1and 1022cm−1is associated with the ordered (or crystalline) and amorphous structures of starch, respectively. Thus, the absorbance ratio of 1047/1022cm−1 (R1047/1022) can be used to quantify the degree of short-range ordering in starch granules. The ratio of 1047/1022 in SLG increased, indicating that debranching enhanced the degree of short-range order of starch molecules. Comparatively SLG were released by debranchingmodification, leading to improved molecular mobility and increased opportunity for molecule alignment with other chains to form new and more stable double helical structure.
Publications
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Progress 04/01/22 to 03/31/23
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?1. Measure cellular permeability of encapsulated quercetin and gingerols and shogaols across Caco-2 cell monolayers. 2. Evaluate gut microbiota-modulating effects of quercetin and ginger extract-loaded starch micro/nanoparticles.
Impacts
What was accomplished under these goals?
1. Preparation of shortlinearglucan(SLG) Waxy maize starch (WMS) was dispersed in acetic acid buffer solution (0.01mol/L, pH 5.5). The starch slurry was cooked and then completely gelatinized. The temperature was adjusted to 50°C and pullulanase (100U/g dry starch) was added. The mixture was incubated at 50°C for 12 h. The mixture was centrifuged and the SLG in the supernatant was precipitated using alcohol at a water/ethanol volume ratio of 1:3. 2. Comparison of pastingpropertiesof WMS and SLG The pasting properties of the samples were measured using a DiscoveryHybridRheometer (DHR-3, TAInstruments) with a starch pasting cell. 3.Rheological properties The DHR-3Rheometer was used to analyze the rheological properties of starch pastes. 4. Preparation of quercetin-loaded SLG micro/nanoparticles The co-precipitation method was used to encapsulate quercetin. The dried shortlinearglucan(SLG) powder (200 mg) was dispersed in 20 mL of distilled water. The solution was cooked and then autoclaved toachievecompletedissolution. 6 mg quercetin was dissolved in 1 mL absolute ethanol, and then added dropwise to 20 mL of the SLG solution under vigorous stirring. The suspensions were continuously stirred until the solvent was completely evaporated to obtain the quercetin-loaded SLG micro/nanoparticles. 5. Preparation of quercetin-loaded SLG/zein complexnanoparticles Quercetin-loaded SLG/zein complexnanoparticles were prepared in thesameway asdescribedabove, except that 6 mg quercetin (Que) and 60 mg zein were first dissolved in 500 µL 80% ethanol and 1 mL absolute ethanol, respectively. After mixing, the Que-Zein dispersions were added dropwise to 20 mL of the SLG solution under vigorous stirring. 6. Determination of encapsulation efficiency (EE) EE was determined as the percentage of quercetin encapsulated in the nanoparticles to the initial amount added. The EE of different SLG nanoparticles ranged from 88.5% to 93.2%. 7.In-vitrogastrointestinal release behavior of the nanoparticles Que-SLG and Que-SLG/zein (or Que-SLG/90G) nanoparticle dispersion was added into a centrifuge tube containing simulated gastric fluid (SGF) release medium (final pepsin Conc. 0.8 mg/ mL, pH 1.5), and incubated in a water bath shaker at 37±2°C for 0.5h. During this process, 0.5mL SGF release medium was withdrawn every 15min. The colloid solution was then mixed with the same volume of simulated intestinal fluid (SIF) release medium (final pancreatin Conc. 5 mg/ mL, porcine bile salt 10 mM, pH 7.0), and then incubated at 37°C for another 4h. At each time interval, sample aliquot was immediately mixed with anhydrous ethanol to inactivate the enzymes. After centrifugation, Que concentration in the supernatant was measured at 373 nm. Results Pasting properties of native WCS and SLG The pasting properties of native WCS and SLG are shown inTable 1. Sharp decreases in the pasting viscosities were observed in SLG. The final viscosity of SLG was very low (31.4 mPa.s), indicating that the SLG starch pastes displayed a liquid-like behavior above 50°C. Table 1. Pasting properties of native waxy corn starch (WCS) and shortlinearglucan (SLG) Sample ID Apparent viscosity Temp(°C) 51.0 66.0 69.1 72.0 92.0 96.0 95.1 95.0 86.0 65.8 50.8 WCS 11.3 28.0241.8 824.6 2933.7 1191.4 2385.8 2725.6 2339.2 2685.3 2711.8 SLG 14.7 14.5 14.4 15.0 20.9 17.4 15.7 14.9 16.6 23.7 31.4 Table 2. Pasting parameters of native waxy corn starch (WCS) and shortlinearglucan (SLG). PV: peak viscosity, TV: trough viscosity, BD: breakdown viscosity, FV: final viscosity, and SB: setback viscosity. PT: pasting temperature. PV (mPa.s) TV (mPa.s)BD (mPa.s)FV (mPa.s)SB (mPa.s) PT (°C) WCS 2933.71191.4 1742.3 2711.8 221.9 69.1 SLG 21.5 16.3 5.2 31.4 -9.9 71.9 Dynamic rheological properties of native waxy corn starch and shortlinearglucan The variation of the storage modulus (G′), loss modulus (G″), and loss tangent (tan δ) as functions of frequency are depicted inTable 3. TheG"of all starch pastes was always higher thanG′, indicating that the viscous component was predominant over the elastic one. TheG′andG″of SLG pastes were significantly lower than those of the native starch, which was consistent with the trends of pasting viscosities. As shown inTable 3, the tan δ values of both native WCS and SLG samples were generally greater than 1, suggesting typicalviscousfluid-likebehavior over the entire frequency range. In addition, tan δ values of both gels were found to be weakly dependent to almost independent of angular frequency. Table 3. Storage modulus (G′), loss modulus (G′′), and loss tangent (tan δ) vs. angular frequency for native and modified starches. WMS: waxy maize starch; SLG: shortlinearglucan. Angular frequency (rad/s) Storage modulus (Pa) Loss modulus (Pa) tanδ WCS SLG WCS SLGWCSSLG 0.628 151.11 58.020 343.935115.763 2.276 1.995 0.791 176.989 53.280 377.862108.113 2.135 2.029 0.996 183.743 49.849 384.916 101.904 2.095 2.044 1.254 198.638 49.011 399.817 100.090 2.013 2.042 1.578 205.803 49.702 402.69997.615 1.957 1.964 1.987 211.106 50.544 403.91496.124 1.913 1.902 2.501 211.787 52.080 398.323 93.794 1.881 1.801 3.149 210.097 50.817 392.433 88.631 1.868 1.744 3.964 206.185 48.900 386.077 81.497 1.872 1.667 4.991 200.478 45.953 383.275 74.753 1.912 1.627 6.283 191.951 42.029 383.014 66.923 1.995 1.592 7.910 182.595 36.874 386.797 58.363 2.118 1.583 9.958 173.495 31.235 394.385 50.184 2.273 1.607 12.537 166.177 26.574 405.995 43.789 2.443 1.648 15.783 161.248 22.713 421.1 39.106 2.612 1.722 19.869 160.697 20.079 441.046 35.514 2.745 1.769 25.014 165.638 18.560 465.732 33.167 2.812 1.787 31.491 177.567 17.343 495.292 31.340 2.789 1.807 39.644 199.664 16.612 528.294 30.162 2.646 1.816 49.909 231.047 16.320 558.933 29.410 2.419 1.802 62.832 269.392 16.132 585.936 28.777 2.175 1.784 5.1 System 1: Que-loaded SLG micro/nanoparticles The accumulative release of Que after incubatedin SGF in the presence of pepsin for 30 min was 44.19%. The accumulative release profile in SIF in the presence of pancreatin is shown inTable 4. In SGF, Que-SLG exhibited a rapid release behavior. Even though SLG cannot be degraded by pepsinin the stomach, Que-SLG nanoparticles seemed to be very sensitive to low pH and released a large amount of Que. On the contrary, a sustained release was observed in SIF. After 4 h incubation, only 48.17 % of Que was released into the medium. Table 4. The accumulative release profile in simulated intestinal fluid (SIF) Time 15 min 30 min 1 h 1.5 h 2 h 3 h 4 h accumulative release (%)26.07 25.29 46.59 39.49 46.59 42.64 48.17 5.2 System 2: Que-loaded SLG/zein andQue-SLG/90Gcomplexnanoparticles To overcome the limitations of earlier release of Que in SGF, two hybridcomplexnanoparticles were prepared: Que-SLG/zein and Que-SLG/90G complexnanoparticles. The accumulative release of Que after incubated in SGF in the presence of pepsin for 30 min was 13.89% and 23.28%, respectively. Due to the compact network formed between SLG and zein (or Phospholipon90G), the majority of Que was retained after digestion in SGF, suggesting that the hybrid nanoparticles had the ability to protect Que from degradation in stomach environment. Table 5. The accumulative release profile in simulated intestinal fluid (SIF) Accumulative release (%)Time 15 min 30 min 1 h 2 h 3 h 4 h Que-SLG/zein 31.27 32.26 33.84 33.93 33.84 34.43 Que-SLG/90 G 26.74 27.62 29.30 29.10 27.23 27.92 The accumulative release profile in SIF in the presence of pancreatin is shown inTable 5. The combinations of SLG with the two polymers also showed a remarkablesustainedrelease effect, only 34.43% and 27.92% of Que was released after digestion in SIF for 4h.
Publications
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Progress 04/01/21 to 03/31/22
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?We will continue with objectives 3 and 4.
Impacts
What was accomplished under these goals?
1. Preparation of modified starch Waxy corn starch and rice were hydrophobically modified through esterification with octenyl succinate anhydride or acetic anhydride. A series of modified starch with different degrees of substitution were prepared by changing the molar ratios of esterifying agents to starch, and varying reaction temperatures and times. 2. Preparation of ginger extract-loaded starch micro/nanoparticles The co-precipitation method was used to encapsulate ginger extract within modified starch. A predetermined amount of modified starch with different degrees of substitution and molar mass was dispersed in deionized water and equilibrated at room temperature for 20 min. Ginger extract was dissolved in ethanol or acetone and added to the modified starchunder vigorous stirring. The suspensions were continuously stirred until the solvent was completely evaporated to obtain the ginger extract-loaded starch micro/nanoparticles. 3. Determination of encapsulation efficiency of ginger extract The encapsulation efficiency (EE) was determined as the percentage of ginger extract encapsulated in the nanoparticles to the initial amount of extract added. The encapsulation efficacy was then calculated using the following equation: EE (%) = (Total amount of ginger extract - Free amount of ginger extract)/ Total amount of ginger extract × 100. The amount of free (unloaded) ginger extract measured using a membrane separation method, using an Amicon Ultra-15 centrifugal filter device (Millipore Corp., Ann Arbor, MI) with 3 kDa molecular weight cut off. Concentrations of ginger compounds were quantitatively analyzed using HPLC-ECD method. The EE of two ginger extract-hydrophobically modified starch nanoparticle was 70.7% and 76.2%, respectively. 4. Glucose release profile of modified starch The digestion of starch was carried out following the Enlgyst method (Englyst et al., 1992). 4.1 Enzyme solution preparation 2 grams of pancreatin (A0585, from porcine pancreas; Sigma) were dispersed in 60 ml of deionized water and stirred for 10 min. The dispersion was centrifuged at 12000g for 10 min. 45 ml of the supernatant were collected and transferred to a beaker. 0.5 ml of amyloglucosidase (A3306, from Aspergillus niger; Sigma) were added to that beaker and the mixture was reserved for later use. 50 ml of a 0.5% dispersion of guar gum were prepared and reserved for later use. 4.2 Modified starch hydrolysis 500 mg of starch (raw and modified) were dispersed in 15 ml of deionized water and allowed to hydrate for two hours. After two hours, 10 ml of the guar gum solution and 10 ml of the enzyme solution were added to the starch. 125 µm aliquots of the starch dispersion were taken at 2, 5, 10, 15, 20, 30, 40, 50, 60, and 120 min and mixed with 1 ml of 80% ethanol previously added in each test tube to ensure the reaction stopped in time. After the hydrolysis, the tubes were centrifuged at 10,000 rpm for 5 minand the supernatants were filtered and transferred to a new tube. 4.3 Glucose release measurements The glucose measurement was carried out using GO Glucose Assay (GAGO20, Sigma). 200ul of GO enzyme and 200 µl of sulfuric acid were added to 100 ul of each digested starch samples. The color of each sample was measured using a 96 well plate spectrophotometer. The absorbance was recorded and used to calculate the amount of glucose in each sample, and the results were normalized to percentage for better comparison. The raw waxy corn starch was hydrolyzed quickly and the release of glucose lasted about 60 min. On the contrary, hydrolysis of encapsulated starch was much slower especially during the first 60 minand a sustained glucose release profile was observed. The digestion kinetics of encapsulated starch could be further tailored through modifying encapsulation layers, e.g., complex coacervation of zein with different types of polysaccharides or thickness of the encapsulation layer. In conclusion, encapsulation of starch granules with zein/polysaccharide provided an effective way to slow down digestion rate of starch. 5. In vitro release study An In vitro release study was carried out using simulated gastric fluid (SGF) with pepsin, and simulated intestinal fluid (SIF) with pancreatin. Briefly, the ginger extract-loaded nanocapsules were first incubated in SGF [an aqueous solution of 1% (w/v) pepsin at pH 1.2]for 30 min. Subsequently, they were separated and incubated in SIF [0.05 M phosphate buffer (pH 7.5) with 1% (w/v) pancreatin present] for 0.5 and 2 h. 5.1 System 1: ginger extract-hydrophobically modified starch nanoparticle 1 The accumulative release of ginger extract after incubated in simulated gastric fluid (SGF) in the presence of pepsin for 30 min was 10.4%. The accumulative release profile in simulated intestinal fluid (SIF) in the presence of pancreatin: Time (h) 0.5 1 2 3 5 24 Release (%) 30.6 44.3 53.2 68.2 75.9 74.3 5.2 System 2: ginger extract-hydrophobically modified starch nanoparticle 2 The accumulative release of ginger extract after incubated in SGF (in the presence of pepsin) for 30 min was 8.6% The accumulative release profile in SIF (in the presence of pancreatin): Time (h) 0.5 1 2 3 5 24 Release (%) 12.8 21.3 34.7 49.9 57.8 55.7 6. Particle size distribution of Zein/alginate encapsulated starch The particle size distribution shows the typical particle size distribution of raw waxy corn starch and the same starch encapsulated with zein and alginate. The encapsulated starch exhibited unimodal and relative narrow size distribution pattern. The number mean diameters of raw and encapsulated waxy corn starch were 8.9µm and 10.8µm, respectively. The slight increase in mean diameter of the encapsulated starch was due to the added encapsulation layer around single starch granule as well as the aggregation of a small portion of the granules. 7. Confocal laser scanning microscopy (CLSM) images of zein/alginate encapsulated waxy corn starch CLSM images of the cross section of waxy corn starch granules showed the morphology of the granules under transmitted and alsothe outer encapsulation layer being covalently labeled with fluorescein-5-isothiocyanate ( FITC). The FITC-labeled encapsulation layer appeared green under CLSM. It could be clearly seen that the starch granules were fully covered by the encapsulation materials.
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