Seokwon Lim

Enhancing Nanoparticle-Based Visible Detection by Controlling the Extent of Aggregation

Visible indication based on the aggregation of colloidal nanoparticles (NPs) is highly advantageous for rapid on-site detection of biological entities, which even untrained persons can perform without specialized instrumentation. However, since the extent of aggregation should exceed a certain minimum threshold to produce visible change, further applications of this conventional method have been hampered by insufficient sensitivity or certain limiting characteristics of the target. Here we report a signal amplification strategy to enhance visible detection by introducing switchable linkers (SLs), which are designed to lose their function to bridge NPs in the presence of target and control the extent of aggregation. By precisely designing the system, considering the quantitative relationship between the functionalized NPs and SLs, highly sensitive and quantitative visible detection is possible. We confirmed the ultrahigh sensitivity of this method by detecting the presence of 20 fM of streptavidin and fewer than 100 CFU/mL of Escherichia coli.

Improving Flavonoid Bioaccessibility using an Edible Oil-Based Lipid Nanoparticle for Oral Delivery

To enhance the oral bioaccessibility of flavonoids, including quercetin, naringenin, and hesperetin, we prepared an edible oil-based lipid nanoparticle (LNP) system. Flavonoid-loaded LNPs were similar to the blank LNP in physicochemical characteristics (z average <154.8 nm, polydispersity index <0.17, and ζ potential < -40.8 mV), and their entrapment efficiency was >81% at 0.3 wt % flavonoid concentration of the lipid phase. In the simulated digestion assay (mouth, stomach, and small intestine), LNPs were hydrolyzed under small intestine conditions and protected successfully incorporated flavonoids (≥94%). Moreover, the relative bioaccessibility of flavonoids was >71%, which was otherwise <15%, although flavonoids were released rapidly from LNPs into the medium. In conclusion, since the flavonoids incorporated in LNPs were preserved well during oral digestion and had improved bioaccessibility, the designed LNP system may serve as an encapsulation strategy to enhance the bioavailability of nonbioaccessible nutraceuticals in foods.

Thermal deactivation kinetics of Pseudomonas fluorescens lipase entrapped in AOT/isooctane reverse micelles.

Thermostability of the lipase (EC 3.1.1.3) was found to be increased by the enzyme-entrapment in 50 mM AOT/isooctane reverse micelles. The half-life (15.75 h) of Pseudomonas fluorescens lipase entrapped in reverse micelles at 70 °C was 9.72- and 11.41-fold longer than those solubilized in a glycerol pool or in 10 mM phosphate buffer (pH 8.0), respectively. The enzyme deactivation model considering a two-step series-type was employed, and deactivation constants for the second step (k₂) at all temperatures were drastically decreased after the lipase was entrapped in reverse micelles. In particular, k₂ (0.0354 h⁻¹) at 70 °C in reverse micelles was 12.33- and 13.14-fold lower than in a glycerol pool or in the phosphate buffer, respectively. The deactivation energies (from k₁, k₂) for the lipase entrapped in the reverse micelles, solubilized in a glycerol pool, or in the aqueous buffer were 7.51, 26.35 kcal/mol, 5.93, 21.08 kcal/mol, and 5.53, 17.57 kcal/mol, respectively.

Bifunctional linker-based immunosensing for rapid and visible detection of bacteria in real matrices

Detection of pathogens present in food and water is essential to help ensure food safety. Among the popular methods for pathogen detection are those based on culture and colony-counting and polymerase chain reaction (PCR). However, the time-consuming nature and/or the need for sophisticated instrumentation of those methods limit their on-site applications. We have developed a rapid and highly sensitive immunosensing method for visible detection of bacteria in real matrices based on the aggregation of AuNPs without requiring any readout device. We use biotinylated anti-bacteria antibodies as bifunctional linkers (BLs) to mediate the aggregation of streptavidin-functionalized gold nanoparticles (st-AuNPs) to produce visually recognizable color change, due to surface plasmon resonance (SPR), which occurs in about 30min of total assay time when the sample is mildly agitated or within three hours in quiescent conditions. The aggregation of st-AuNPs, which produces the indication signal, is achieved very differently than in visual detection methods reported previously and hence affords ultrahigh sensitivity. While BLs can both bind to the target and crosslink st-AuNPs, their latter function is essentially disabled when they bind to the target bacteria. By varying the amount of st-AuNPs used, we can tailor the assay effectiveness improving limit of detection (LOD) down to 10CFUmL-1 of E. coli and Salmonella. Test results obtained with tap water, lake water and milk samples show that assay performance is unaffected by matrix effects. Further, in a mixture of live and autoclaved E. coli cells our assay could detect only live cells. Therefore, our BL-based immunosensor is suitable for highly sensitive, rapid, and on-site detection of bacteria in real matrices.

Enhancing the Stability of Lipid Nanoparticle Systems by Sonication during the Cooling Step and Controlling the Liquid Oil Content

Aggregation of unstable particles in water limits the application of lipid nanoparticle (LNP) systems to foods despite the capability to encapsulate lipophilic bioactive components. This study exploits a preparation process that can reduce the aggregation of LNPs. Sonication during the cooling step (postsonication) for 4, 5, or 6 min was applied to increase the covering effect of Tween 20 on the particle. Additionally, LNPs were prepared using fully hydrogenated canola oil (FHCO) blended with 0-30 wt % liquid canola oil (LCO) of the lipid phase. Surfactant surface load data indicate that the postsonication might make nonemulsifying Tween 20 diffuse from the aqueous phase to droplet surfaces, which could decrease the crystallinity index (CI) of LNPs due to the inhibition of lipid crystallization. Moreover, the LCO content in lipid matrix could decrease the CI, which could reduce the formation of hydrophobic patches on the particle surface. Therefore, the postsonication and the LCO addition in the matrix could effectively prevent aggregation among hydrophobic patches. This improved colloidal stability of LNPs was verified by the particle shape in transmission electron microscopy and the gelation test. Consequently, LNPs fabricated using 6 min postsonication and 30 wt % LCO in the lipid exhibited the greatest stability (size, 202.3 nm; CI, 57.5%; Tween 20 surface load, 10.29 mg m(-2)). This study may serve as a basis for further research that aims to develop delivery systems for functional foods.

Optimization of Enzymatic Treatment for Compound K Production from White Ginseng Extract by Response Surface Methodology

Ginsenoside 20-O-β-D glucopyranosyl-20(S)-protopanaxadiol (compound K), a minor ginsenoside, is not found in white raw ginseng, but has better bioavailability than the major ginsenosides in ginseng. Employing commercial enzyme packages for industrial applications, the optimum conditions for enzymatic transformation for the highest content of compound K was explored to enhance the health benefits of ginseng extract. Cytolase PCL 5 was selected from commercial enzyme packages nominated for high β-glucosidase activity. By response surface methodology, the optimal conditions were identified as 78 h of treatment at pH 4.3 at 55.4 °C for 2.068 mg/mL of compound K, showing good agreement with the experimental value.

The possible presence of natural β-D-glucosidase inhibitors in jujube leaf extract.

Isoquercitrin is a phenolic compound well-known for having greater health benefits than quercitin, its aglycone derivative, and other related glycosides. However, isoquercitrin is rarely found in nature. Here, we optimized the conditions for the enzymatic transformation of isoquercitrin from rutin that was extracted from jujube leaf using the hesperidinase, enzyme complex containing β-D-glucosidase and α-L-rhamnosidase. The maximum productivity (2.57±0.16mg/mL) was experimentally found under the following conditions: 47.3°C, 52.16h, and pH 5.31, which agreed well with the predicted value (2.65mg/mL). However, the achievement of this maximum yield was due to the absence of β-D-glucosidase activity. Further investigations using a β-D-glucosidase assay and reaction measurements under various conditions revealed that the β-D-glucosidase activity was not blocked by denaturation or known inhibitory factors. Currently, there are no recognized β-D-glucosidase inhibitors present in the jujube leaf; however, our observations strongly suggest that an unidentified β-D-glucosidase inhibitor exists in jujube leaf extract.

Control of the gastrointestinal digestion of solid lipid nanoparticles using PEGylated emulsifiers

We prepared solid lipid nanoparticles (SLNs) with tristearin and various emulsifiers which had different chain length PEGs (10-100 times-repetition of ethylene glycol) to control their digestion fate in the gastrointestinal tract. Fabricated SLNs after acidic/high-ionic-strength media treatment were stable regardless of the ζ-potential (ZP) disappearance. Additionally, highly PEGylated SLNs successfully hindered the adsorption of both bile acid (BA) and lipase on the SLN surface, while lowly PEGylated SLNs interrupted that of only lipase. In simulated small intestinal fluid, lipolysis of highly PEGylated SLNs increased with decrease of the emulsifier density on the SLNs, whereas lipolysis of lowly PEGylated SLNs increased with decrease of the particle size. These results suggested that high PEGylation was more efficient than low PEGylation to hinder the lipolysis initiated from the competitive replacement of the SLN-covering emulsifiers with BAs. Consequently, the SLN digestion could be controlled by choosing the length and concentration of PEGylated emulsifiers.

Optimization of Acetic Acid Fermentation for Producing Vinegar from Extract of Jujube (Zizyphus jujuba Mill.) Fruits

The optimum conditions for producing vinegar from Jujube (ziziphus jujuba) juice using Acetobacter aceti were exploited by employing the response surface methodology (RSM). In addition to the initial concentration of ethanol, which is known to be a significant factor affecting acetic acid fermentation, the effects of initial concentration of Jujube juice, A. aceti concentration, pH, and temperature on acetic acid fermentation were also investigated. Out of these factors, the effects of the initial concentration of jujube juice and inoculation amount of A. aceti were determined to be negligible based on statistical analysis. By employing the face-centered experimental design in RSM, the optimum conditions for acetic acid fermentation were exploited for achieving maximum acidity and acetic acid production. The coefficients (R 2 ) of the derived equations from the response surface regression were 0.71 and 0.78 for acidity and acetic acid production, respectively. The maximum production of acetic acid was expected to be 52.76 mg/mL from 25% jujube extract at 21.75 o C with 7.69% alcohol content.

Gas-sensing array application for on-line monitoring in a heat-responsive bioprocess of Streptomyces griseus HUT 6037

The stress-responsive bioprocess concept has been developed into an environmentally friendly biosensor with low energy consumption and high recovery yield. To improve chitosanase production during Streptomyces griseus cultivation, heat-stress was applied to the bioprocess with monitoring using an on-line gas sensing system. When exposed to heat-stress with liposomes, the chitosanase productivity was 2.6 times greater than for conventional cultivation. The mixed gas components could be distinguished using a principal component analysis during chitosanase production. The online-monitoring system reflected basic changes in growth conditions and metabolite formation in cells. The array type gas-sensing system was capable of detecting bacterial infection faster than conventional sensor systems. The gas sensor system can play a key role in monitoring and controlling stress levels in a stress-responsive bioprocess.