Microencapsulation of endophytic LAB (KCC-41) and its probiotic and fermentative potential for cabbage kimchi.

The aim of the present study was to isolate novel lactic acid bacteria (LAB) from hairy vetch forage crop and characterize their probiotic and fermentative potential for preparing Korean cabbage kimchi. First, functional characterization of isolated strains such as antagonistic property, auto-aggregation, antibiotic susceptibility, and extracellular enzyme production was performed. The isolated Lactobacillus plantarum KCC-41 strain was able to inhibit pathogenic fungal spore formation. It showed susceptibility to common commercial antibiotics drugs. The selected LAB strain was then subjected to microencapsulation with alginate biopolymer. Its ability to survive in in vitro simulated gastro-intestinal fluid was evaluated. It was also used in the fermentation of cabbage kimchi samples. The encapsulated KCC-41 strain could effectively lead to kimchi fermentation in terms of reducing its pH and dominating bacterial count. It also significantly increased organic acid production than non-encapsulated LAB (KCC-41) for cabbage kimchi samples.

Potential Sustainable Properties of Microencapsulated Endophytic Lactic Acid Bacteria (KCC-42) in In-Vitro Simulated Gastrointestinal Juices and Their Fermentation Quality of Radish Kimchi.

The objective of this study was to investigate alginate microencapsulated lactic acid bacteria (LAB) fermentation quality of radish kimchi sample and its potential survivability in different acidic and alkaline environments. Initially, we isolated 45 LAB strains. One of them showed fast growth pattern with potential probiotic and antifungal activities against Aspergillus flavus with a zone of inhibition calculated with 10, 8, 4mm for the 4th, 5th, and 6th day, respectively. Therefore, this strain (KCC-42) was chosen for microencapsulation with alginate biopolymer. It showed potential survivability in in-vitro simulated gastrointestinal fluid and radish kimchi fermentation medium. The survival rate of this free and encapsulated LAB KCC-42 was 6.85 × 105 and 7.48× 105 CFU/ml, respectively; the viability count was significantly higher than nonencapsulated LAB in simulated gastrointestinal juices (acid, bile, and pancreatin) and under radish kimchi fermentation environment. Kimchi sample added with this encapsulated LAB showed increased production of organic acids compared to nonencapsulated LAB sample. Also, the organic acids such as lactic acid, acetic acid, propionic acid, and succinic acid production in fermented kimchi were measured 59mM, 26mM, 14mM, and 0.6mM of g/DW, respectively. The production of metabolites such as lactic acid, acetic acid, and succinic acid and the bacteria population was high in microencapsulated LAB samples compared with free bacteria added kimchi sample. Results of this study indicate that microencapsulated LAB KCC-42 might be a useful strategy to develop products for food and healthcare industries.

Quantification of major phenolic and flavonoid markers in forage crop Lolium multiflorum using HPLC-DAD

ABSTRACT The objective of this study was to perform preliminary screening of phytochemical compounds and quantification of major phenolics and flavonoid markers in Italian ryegrass extract using HPLC-DAD. Previously, LC-MS analysis has identified different phenolic acids, including caffeic acid, ferulic acid, p-coumaric acid, chlorogenic acid, dihydroxy benzoic acid, propyl gallate, catechin, and six flavonoids including rutin hydroxide, luteolin, kaemferol, vitexin, narcissoside, and myricetin from Italian ryegrass extract. In the present study, Italian ryegrass silage powder was extracted with ethanol: water for 20 min at 90 °C. The extract targeted optimum yield of phenolic acids and flavonoids. Crude phenolic acid and flavonoids were then purified by solid phase extraction method. Purified fractions were then injected into HPLC with a diode-array detector. Quantified concentrations of isolated phenolic acids and flavonoids ranged from 125 to 220 µg/g dry weight. Limits of detection and limits of quantification for all standards (unknown compounds) ranged from 0.38 to 1.71 and 0.48 to 5.19 µg/g dry weight, respectively. Obtained values were compared with previous literatures, indicating that our HPLC-DAD quantification method showed more sensitivity. This method showed better speed, accuracy, and effectiveness compared to previous reports. Furthermore, this study could be very useful for developing phenolic acids and flavonoids from compositions in Italian ryegrass silage feed for pharmaceutical applications and ruminant animals in livestock industries.

Paclitaxel-incorporated nanoparticles of hydrophobized polysaccharide and their antitumor activity.

The aim of this study was to characterize paclitaxel-incorporated polysaccharide nanoparticles and evaluate their antitumor activity in vitro and in vivo. Pullulan was hydrophobically modified using acetic anhydride to make the paclitaxel-incorporated nanoparticles. Pullulan acetate (PA) was used to encapsulate paclitaxel using the nanoprecipitation method. The particles had spherical shapes under electron microscopy with sizes <100 nm. The sizes of paclitaxel-incorporated nanoparticles increased to >100 nm, and higher drug feeding induced higher particle size and drug content. Initial drug burst release was observed until 2 days and then the drug was continuously released over 1 week. Intrinsic cytotoxicity of empty PA nanoparticles was tested with RAW264.7 macrophage cells for biocompatibilty. The viability of RAW264.7 cells was >93% at all concentrations of empty PA nanoparticles, indicating that the PA nanoparticles are not acutely cytotoxic to normal human cells. The nanoparticles showed lower antitumor activity in vitro against HCT116 human colon carcinoma cells than that of paclitaxel itself, indicating the sustained release properties of nanoparticles. An in vivo study using HCT116 human colon carcinoma-bearing mice showed that paclitaxel-incorporated PA nanoparticles reduced tumor growth more than that of paclitaxel itself. These results indicate that PA paclitaxel-incorporated nanoparticles are a promising candidate for antitumor drug delivery.

Antitumor effect of adriamycin-encapsulated nanoparticles of poly(DL-lactide-co-glycolide)-grafted dextran.

In this study, we prepared adriamycin (ADR)-encapsulated core-shell type nanoparticles of a poly(DL-lactide-co-glycolide) (PLGA) grafted-dextran (DexLG) copolymer and evaluated its antitumor activity in vitro and in vivo. The particle size of ADR-encapsulated DexLG nanoparticles was around 50-200 nm and the morphology was spherical shapes at transmission electron microscopy (TEM) observation. Since reconstitution of lyophilized nanoparticles is essential to practical use in vivo, ADR-encapsulated DexLG nanoparticles were lyophilized and reconstituted them into deionized water. Although reconstitution process caused increase of particle size, drug release behavior of nanoparticles was not significantly changed before and after reconstitution process. The ADR-encapsulated DexLG nanoparticles were less cytotoxic than free ADR plus empty nanoparticles at in vitro, while empty DexLG nanoparticles did not significantly affect cell viability. Even if free ADR plus empty nanoparticles are most effective to inhibit tumor growth at tumor-induced animal model using CT-26 cells, ADR-encapsulated DexLG nanoparticles showed increased survivability of mice. These results indicated that ADR-encapsulated DexLG nanoparticles are promising vehicles for antitumor drug delivery.

Cytotoxicity of amphotericin B-incorporated polymeric micelles composed of poly(DL-lactide-co-glycolide)/dextran graft copolymer.

In this study, we prepared amphotericin B (AmpB)-encapsulated polymeric micelle of poly(DL-lactideco-glycolide) (PLGA) grafted-dextran (DexLG) copolymer for the cytotoxicity test. The average particle size of AmpB-encapsulated DexLG polymeric micelles was around 30 approximately 70 nm and their morphology showed spherical shapes. Since aggregation states of AmpB are related to intrinsic cytotoxicity, prevention of AmpB aggregation in aqueous solution will provide low cytotoxicity and increased antimicrobial activity for the infectious disease. At UV/VIS spectrum measurement, polymeric micelle prepared from methanol/water mixture (method B) showed a monomeric state of AmpB while polymeric micelle prepared from DMSO (method A) showed an aggregated state. During the hemolysis activity test, polymeric micelle from method B showed reduced hemolysis activity compared to AmpB itself and polymeric micelle from method A. These results indicated that AmpB-incorporated polymeric micelle prepared from methanol/water mixture has low cytotoxicity and favorable antimicrobial activity.

Amphotericin B-incorporated polymeric micelles composed of poly(d,l-lactide-co-glycolide)/dextran graft copolymer.

In this study, we prepared amphotericin B (AmpB)-encapsulated polymeric micelle of poly(d,l-lactide-co-glycolide) (PLGA) grafted-dextran (DexLG) copolymer and characterized its physicochemical properties in vitro. The average particle size of AmpB-encpasulated DexLG polymeric micelles was around 30-150nm while particle size of empty polymeric micelles was below 100nm according to the copolymer composition. The morphology of AmpB-encapsulated polymeric micelle of DexLG copolymer was spherical shapes at transmission electron microscopy (TEM) observation. At 1H NMR study, specific peaks of AmpB and DexLG copolymer was obtained at DMSO but specific peaks characterized to AmpB and PLGA was disappeared at D2O environment. These results indicated that AmpB was encapsulated into the micellar core of polymeric micelle. XRD results also support these results, indicating that specific crystal peaks of AmpB and broad peaks of DexLG copolymer were obtained but specific peaks of AmpB was disappeared at polymeric micelles while physical mixture of AmpB/empty polymeric micelles showed both specific peaks. Drug release rate was decreased according to the increase of drug contents and increase of PLGA component of DexLG copolymer. At the minimal inhibition concentration (MIC) study using Candida albicans, AmpB-encapsulated polymeric micelle showed almost similar effectives on the growth inhibition of microorganisms. These showed that AmpB-encapsulated polymeric micelle of DexLG copolymer can be considered to potential antifungal agent carriers.

Doxorubicin release from core-shell type nanoparticles of poly(DL-lactide-co-glycolide)-grafted dextran.

In this study, we prepared core-shell type nanoparticles of a poly(DL-lactide-co-glycolide) (PLGA) grafted-dextran (DexLG) copolymer with varying graft ratio of PLGA. The synthesis of the DexLG copolymer was confirmed by 1H nuclear magnetic resonance (NMR) spectroscopy. The DexLG copolymer was able to form nanoparticles in water by self-aggregating process, and their particle size was around 50 nm approximately 300 nm according to the graft ratio of PLGA. Morphological observations using a transmission electron microscope (TEM) showed that the nanoparticles of the DexLG copolymer have uniformly spherical shapes. From fluorescence probe study using pyrene as a hydrophobic probe, critical association concentration (CAC) values determined from the fluorescence excitation spectra were increased as increase of DS of PLGA. 1H-NMR spectroscopy using D2O and DMSO approved that DexLG nanoparticles have core-shell structure, i.e. hydrophobic block PLGA consisted inner-core as a drug-incorporating domain and dextran consisted as a hydrated outershell. Drug release rate from DexLG nano-particles became faster in the presence of dextranase in spite of the release rate not being significantly changed at high graft ratio of PLGA. Core-shell type nanoparticles of DexLG copolymer can be used as a colonic drug carrier. In conclusion, size, morphology, and molecular structure of DexLG nanoparticles are available to consider as an oral drug targeting nanoparticles.

Adriamycin release from self-assembling nanospheres of poly(DL-lactide-co-glycolide)-grafted pullulan.

Poly(DL-lactide-co-glycolide)-graft pullulan (PuLG) was synthesized to produce a hydrophobically modified polysaccharide. Specific pullulan and poly(DL-lactide-co-glycolide) (PLGA) (abbreviated as PuLG) appeared in the peaks of the PuLG spectra on (1)H NMR spectroscopy, suggesting that PLGA was successively grafted to the pullulan backbone. PuLG nanospheres have a round shape with a particle size of about 75-150 nm. From the fluorescence excitation spectra in a fluorescence probe study, the critical association concentration (CAC) values were determined to be 0.017 g/l for PuLG-1, 0.0054 g/l for PuLG-2, and 0.0047 g/l for PuLG-3. The drug contents of the PuLG nanospheres were approximately 20-30% (w/w). As the drug contents of PuLG nanospheres increased, the drug release rate from nanospheres decreased. The drug release rate from PuLG nanospheres was delayed as the molecular weight of PuLG increased. PuLG copolymer with higher graft ratio of PLGA showed slower degradation rate rather than that with lower graft ratio. Since degradation rate of PuLG was taken over 1 month, drug release was governed by diffusion mechanism rather than degradation mechanism.