Galactose-Containing Polymer-DOX Conjugates for Targeting Drug Delivery

A novel multifunctional drug delivery system was fabricated by conjugating galactose-based polymer, methoxy-poly(ethylene glycol)-block-poly(6-O-methacryloyl-D-galactopyranose) (mPEG-b-PMAGP) with doxorubicin (DOX) via an acid-labile carbamate linkage. The mPEG-b-PMAGP-co-DOX nanoparticles were spherical in shape, and the diameter determined by dynamic light scattering (DLS) was 54.84?±?0.58 nm, larger than that characterized by transmission electron microscopy (TEM). The in vitro drug release profiles were studied, and the release of DOX from the nanoparticles was pH-responsive. The cellular uptake behavior of free-DOX and mPEG-b-PMAGP-co-DOX nanoparticles by asialoglycoprotein (ASGP) receptor-positive cancer cell line (HepG2) and ASGP receptor-negative cancer cell lines (MCF-7 and A549 cells) was evaluated by confocal laser scanning microscopy (CLSM) and flow cytometry (FCM), respectively. The mPEG-b-PMAGP-co-DOX nanoparticles which contain galactose functional groups exhibited higher cellular uptake behavior via ASGP receptor-mediated endocytosis in HepG2 cells than in other two cancer cells. The in vitro cytotoxicity assay manifested that the mPEG-b-PMAGP-co-DOX nanoparticles exhibited higher anticancer efficacy against HepG2 cells than MCF-7 cells. These results indicated that the multifunctional mPEG-b-PMAGP-co-DOX nanoparticles possessing pH-responsible and hepatoma-targeting function have great potential to be used as a targeting drug delivery system for hepatoma therapy.     

High alkaline activity of a thermostable α-amylase (cyclomaltodextrinase) from thermoacidophilic <Emphasis Type="Italic">Alicyclobacillus</Emphasis> isolate

It is well demonstrated that glycosyl hydrolases from Alicyclobacillus strains are general thermoacidophilic enzymes and are ideal proteins for industrial applications. In this study, a thermophilic Alicyclobacillus α-amylase of glycoside hydrolases 13_20 subfamily, AMY1, was identified from an Alicyclobacillus strain and efficiently expressed in the host Escherichia coli BL21 CodonPlus. In agreement with other reported Alicyclobacillus hydrolases, the purified AmyY1 had an optimal pH of 6.0–6.5 in phosphate or citrate/Na₂HPO₄ buffers, and a remarkably decreased activity at pH 8.0. Differently, much higher activity was detected in the alkaline glycine/NaOH reaction mixtures. Compared to the highest amylolytic activity at pH 6.0, AmyY1 exhibited 230 and 116% activities at pH 8.0 and 9.0, respectively. This glycine-activation was further confirmed by a supplementation of glycine into the assay mixtures. During the digestions of various raw starches, AmyY1 also exhibited high hydrolysis efficiency under acidic or alkaline conditions. Findings in this study not only endow AMY1 with much broad applications, but also may provide a novel field for the application potentials of some other Alicyclobacillus hydrolases.     

Design and Evaluation of Hydrophilic Matrix System for pH-Independent Sustained Release of Weakly Acidic Poorly Soluble Drug

The aim of this research was to design and evaluate a hydrophilic matrix system for sustained release of glipizide, a weakly acidic poor soluble drug. A combination of inclusion complexation and microenvironmental pH modification techniques was utilized to improve the dissolution and pH-independent release of glipizide. Hydroxypropyl-β-cyclodextrin (HP-β-CD) was used as the complexation agent while sodium citrate and magnesium oxide (MgO) were used as model pH modifiers. The hydrophilic matrix tablets were prepared by powder direct compression and evaluated by in vitro dissolution study respectively in pH 6.8 and pH 1.2 dissolution media. The formulations containing MgO exhibited increased cumulative drug release from less than 40% in the reference formulation to 90% within 24 h in acidic media (pH 1.2). The release profile in acidic media was similar to the alkaline media (pH 6.8) with a similarity factor (f₂) of 55.0, suggesting the weakening of the effect of pH on the dissolution efficiency of glipizide. The release profile fitted well into the Higuchi model and the dominant mechanism of drug release was Fickian diffusion while case II transport/polymer relaxation occurred. In conclusion, combining inclusion complexation agents and pH modifiers had improved the dissolution of glipizide as well as achieved the pH-independent release profile.     

Comparative Evaluation of Nimesulide-Loaded Nanoparticles for Anticancer Activity Against Breast Cancer Cells

Recent clinical and epidemiological researches have declared that non-steroidal anti-inflammatory agents may display as antineoplastic agents and indicate pro-apoptotic and antiproliferative effects on cancer cells. The major purpose of this research was to develop a novel poly(ethyleneglycol)-block-poly(ε-caprolactone) (PEG-b-PCL) nano-sized particles encapsulated with nimesulide (NMS), a selective COX-2 inhibitor, and to evaluate its anticancer activity against MCF-7 breast cancer cells. NMS-encapsulated PEG-b-PCL nanoparticles were fabricated using three different production techniques: (i) by emulsion-solvent evaporation using a high shear homogenizer, (ii) by emulsion-solvent evaporation using an ultrasonicator, and (iii) by nanoprecipitation. Nanoparticles were evaluated with respect to the entrapment efficiency, size characteristics, drug release rates, thermal behavior, cell viability assays, and apoptosis. The resulting nanoparticles were found to be spherical shapes with negative surface charges. The average diameter of all nanoparticles ranged between 148.5 and 307.2 nm. In vitro release profiles showed that all nanoparticles exhibited a biphasic release pattern. NMS-loaded PEG-b-PCL nanoparticles demonstrated significant anticancer activity against MCF-7 breast cancer cells in a dose-dependent manner, and the effects of nanoparticles on cell proliferation were significantly affected by the preparation techniques. The nanoparticles developed in this work displayed higher potential for the NMS delivery against breast cancer treatment for the future.     

Properties of Probiotics <Emphasis Type="Italic">Kocuria</Emphasis> SM1 and <Emphasis Type="Italic">Rhodococcus</Emphasis> SM2 Isolated from Fish Guts

This study characterized probiotics Kocuria SM1 and Rhodococcus SM2, which were recovered from the intestinal microbiota of rainbow trout (Oncorhynchus mykiss, Walbaum). The cultures were Gram-positive, non-motile, catalase-positive and oxidase-negative cocci or rods. Cell multiplication of SM1 and SM2 was observed at 4–37 °C (45 °C for SM1), in 0–20% (w/v) NaCl and at pH 2–11. The viability was not affected when exposed to pepsin at pH 2.0 and 3.0, and pancreatin at pH 8.0. Neither isolates were chrome azurol S-positive for siderophore production. Of the 19 common enzymes analysed using the API-ZYM system, only 8 were evident in the culture of SM1 compared to 11 enzymes for SM2. The secondary metabolites of both probiotics were inhibitory to Acinetobacter baumannii, Vibrio anguillarum and V. ordalii; SM2 inhibited Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Staphylococcus aureus. SM2 was resistant to penicillin and sulphatriad, out of six antimicrobial agents; SM1 was resistant to sulphatriad. These results suggest that Kocuria SM1 and Rhodococcus SM2 are able to grow over a wide range of temperature, salinity and pH, including in conditions that mimic the gastrointestinal environment of fish and produce extracellular enzymes that may have a role in the host digestive processes. Importantly, Rhodococcus SM2 displays a high degree of bacteriocinogenic potential against multi-drug-resistant human pathogens that have never been documented among the gut microbiota of fish.     

Antifungal Activity of Chitosan-Coated Poly(lactic-co-glycolic) Acid Nanoparticles Containing Amphotericin B

Amphotericin B (AmB) is one of the most used drugs for the treatment of systemic fungal infections; however, the treatment causes several toxic manifestations, including nephrotoxicity and hemolytic anemia. Chitosan-coated poly(lactide-co-glycolide) (PLGA) nanoparticles containing AmB were developed with the aim to decrease AmB toxicity and propose the oral route for AmB delivery. In this work, the antifungal efficacy of chitosan-coated PLGA nanoparticles containing AmB was evaluated in 20 strains of fungus isolates from patients with vulvovaginal candidiasis (01 Candida glabrata and 03 Candida albicans), bloodstream infections (04 C. albicans and 01 C. tropicalis) and patients with urinary tract infection (04 Candida albicans, 02 Trichosporon asahii, 01 C. guilhermondii, 03 C. glabrata) and 01 Candida albicans ATCC 90028. Moreover, the cytotoxicity over erythrocytes was evaluated. The single-emulsion solvent evaporation method was suitable for obtaining chitosan-coated PGLA nanoparticles containing AmB. Nanoparticles were spherical in shape, presented mean particle size about 460 nm, positive zeta potential and encapsulation efficiency of 42%. Moreover, nanoparticles prolonged the AmB release. All the strains were susceptible to plain AmB and nanostructured AmB, according to EUCAST breakpoint version 8.1 (resistant > 1 μg/mL), using broth microdilution method. In C. albicans (urine, blood, and vulvovaginal secretion isolates, and 1 ATCC), the MIC value of AmB-loaded nanoparticles varied from 0.25 to 0.5 μg/mL and EUCAST varied from 0.03 to 0.5 μg/mL. In urine and vulvovaginal secretion isolates of C. glabrata, the MIC value of AmB-loaded nanoparticles varied from 0.25 to 0.5 μg/mL and EUCAST varied from 0.03 to 0.015 μg/mL. In urine isolates of C. guilhermondii, the MIC value of AmB-loaded nanoparticles was 0.12 μg/mL and EUCAST was 0.06 μg/mL. In blood isolates of C. tropicalis, the MIC value of AmB-loaded nanoparticles was 0.5 μg/mL and EUCAST was 0.25 μg/mL. Finally, in urine isolates of T asahii, the MIC value of AmB-loaded nanoparticles was 1 μg/mL and EUCAST varied from 0.5 to 1 μg/mL. In the cytotoxicity assay, plain AmB was highly hemolytic (100% in 24 h) while AmB-loaded chitosan/PLGA nanoparticles presented negligible hemolysis.     

Lectin-Conjugated Clarithromycin and Acetohydroxamic Acid-Loaded PLGA Nanoparticles: a Novel Approach for Effective Treatment of <Emphasis Type="Italic">H. pylori</Emphasis>

Helicobacter pylori infection remains challenging as it mainly colonized beneath the deep gastric mucosa and adheres to epithelial cells of the stomach. Concanavalin-A (Con-A)-conjugated gastro-retentive poly (lactic-co-glycolic acid) (PLGA) nanoparticles of acetohydroxamic acid (AHA) and clarithromycin (CLR) were prepared and evaluated under in vitro conditions. Solvent evaporation method was employed for preparation of nanoparticles and characterized for particle size distribution, surface morphology, percent drug entrapment, and in vitro drug release in simulated gastric fluid. Optimized nanoparticles were conjugated with Con-A and further characterized for Con-A conjugation efficiency and mucoadhesion and tested for in vitro anti-H. pylori activity. The conjugation with Con-A further sustained the drug release over a period of 8 h when compared to non-conjugated nanoparticles of AHA and CLR. In vitro anti H. pylori study confirmed that Con-A-conjugated nanoparticles containing both drugs, i.e., CLR and AHA, had shown maximum zone of inhibition compared to other formulations. In a nut shell, results suggest that the developed systems could be used for better therapeutic activity against H. pylori infection.     

Enhancement of thiamine release during synthetic mutualism between <Emphasis Type="Italic">Chlorella sorokiniana</Emphasis> and <Emphasis Type="Italic">Azospirillum brasilense</Emphasis> growing under stress conditions

Thiamine release during synthetic mutualism between Chlorella sorokiniana co-immobilized in alginate beads with the microalgae growth-promoting bacterium Azospirillum brasilense was measured under stress conditions of pH, light intensity, and nitrogen starvation in short-term experiments. Thiamine release in the co-immobilized treatment was significantly higher at acidic pH compared to thiamine released by either microorganism alone. Under slightly alkaline pH, C. sorokiniana released the highest amount of thiamine. At stressful pH 6, the co-immobilized treatment released a higher quantity of thiamine than the sum of thiamine released by either microorganisms when immobilized separately. Release of thiamine by C. sorokiniana alone or co-immobilized was light intensity dependent; with higher the light intensity, more thiamine was released. Extreme light intensity negatively affected growth of the microalgae and release of thiamine. Nitrogen starvation during the first 24 h of culturing negatively affected release of thiamine by both microorganisms, where C. sorokiniana was more severely affected. Partial or continuous nitrogen starvation had similar negative effects on C. sorokiniana, but co-immobilization improved thiamine release. These results indicate that thiamine is released during synthetic mutualism between C. sorokiniana and A. brasilense, and this happens specifically during the alleviation of pH stress in the microalgae.     

Colon Delivery of Budesonide: Evaluation of Chitosan–Chondroitin Sulfate Interpolymer Complex

The present study was aimed at formulating tablets comprising of coating susceptible to microbial enzyme degradation for releasing budesonide in the colon. Tablets prepared by using Avicel® pH 102 as diluent and Eudragit® L100-55 as binder were coated to a weight gain of 10% w/w employing aqueous mixtures containing chitosan (CH) and chondroitin sulfate (CS). The interpolymer complex between CH and CS was characterized using Fourier transform infrared (FTIR) and differential scanning calorimetery (DSC) studies. The tablets were evaluated for release of budesonide through in vitro in vivo studies. Formation of bonds between –COO^? and –OSO ₃ ^? groups of CS and –NH ₃ ⁺ groups of CH was evident in the FTIR spectra of these interpolymer complexed (IPC) films. The DSC thermograms of these films revealed one endothermic transition between 190°C and 205°C, suggesting the formation of new bonds in the IPC. The pH sensitive swelling exhibited by these films was observed to be a function of CH concentration. Tablets coated with aqueous mixtures containing 40:60 or 50:50 ratio of CH/CS totally prevented the release of budesonide in pH 1.2 buffer. The peaks (FTIR) and endothermic transitions (DSC) characteristic of interpolymer complexation were observed to remain unaffected after sequential exposure of the films to pH 1.2 and pH 7.4 buffer IP. This proved the versatility of these IPC films for colon delivery. C _max of 1,168.99 and 1,174.2 ng/mL, respectively, at 12 and 8 h post-oral dosing of tablets coated with 40:60 or 50:50 ratio of CH/CS was observed in rats. The aqueous CH/CS (40:60) coating could provide a facile method for delivering budesonide to the colon.     

Aciduric Strains of <Emphasis Type="Italic">Lactobacillus reuteri</Emphasis> and <Emphasis Type="Italic">Lactobacillus rhamnosus</Emphasis>, Isolated from Human Feces,Have Strong Adhesion and Aggregation Properties

Human feces were streaked onto MRS Agar adjusted to pH 2.5, 3.0, and 6.4, respectively, and medium supplemented with 1.0% (w/v) bile salts. Two aciduric strains, identified as Lactobacillus reuteri HFI-LD5 and Lactobacillus rhamnosus HFI-K2 (based on 16S rDNA and recA sequences), were non-hemolytic and did not hydrolyze mucin. The surface of Lactobacillus reuteri HFI-LD5 cells has a weak negative charge, whereas Lactobacillus rhamnosus HFI-K2 has acidic and basic properties, and produces exopolysaccharides (EPS). None of the strains produce bacteriocins. Both strains are resistant to several antibiotics, including sulfamethoxazole-trimethoprim and sulphonamides. The ability of Lactobacillus reuteri HFI-LD5 and Lactobacillus rhamnosus HFI-K2 to grow at pH 2.5 suggests that they will survive passage through the stomach. EPS production may assist in binding to intestinal mucus, especially in the small intestinal tract, protect epithelial cells, and stimulate the immune system. Lactobacillus reuteri HFI-LD5 and Lactobacillus rhamnosus HFI-K2 may be used as probiotics, especially in the treatment of small intestinal bacterial overgrowth (SIBO).     

Early Stage HIV Management and Reduction of Stavudine-Induced Hepatotoxicity in Rats by Experimentally Developed Biodegradable Nanoparticles

The objectives of this research work were to develop optimized nanoparticulate formulations of poly (d,l-lactic-co-glycolic acid) (PLGA) (85:15) with an anti-AIDS drug stavudine and to evaluate their in-vitro uptake by the macrophages and hepatotoxicity in-vivo. Nanoparticles were prepared by nanoprecipitation method based on a factorial design with varying parameters such as the amounts of polymer and stabilizer used. Physicochemical characterizations such as drug–excipient interaction, surface morphology, particle size, and zeta potential measurements were carried out. The best formulation was selected and tagged with fluorescein isothiocyanate (FITC) for cellular uptake study of the formulation. In-vitro uptake of nanoparticles by macrophages was carried out. Formulation-induced hepatotoxicity was assessed by analyzing some serum hepatotoxic parameters and hepatic histology following 10-day treatment in comparison with the free drug. Nanoparticles exhibited smooth surface with particle size 84–238 nm, high entrapment efficiency (approx 85%), and negative surface charge. Formulations showed a sustained drug release pattern over the study period. In-vitro uptake study by macrophages exhibited a time-dependent profile. In-vivo studies on rats showed improvement in the serum parameters and maintenance of the integrity of the hepatic architecture indicating decreased hepatotoxicity with the formulations as compared to the free drug. The experimental results showed a positive outcome in the development of antiretroviral drug carrier exhibiting sustained drug release, macrophage-targeted delivery characteristics, and having reduced hepatoxicity. This could be beneficial for the management of early stage of HIV infection besides reducing the drug load for effective treatment, thereby offering an attractive option in AIDS therapy.     

The ectomycorrhizae of <Emphasis Type="Italic">Lactarius rimosellus</Emphasis> and <Emphasis Type="Italic">Lactarius acatlanensis</Emphasis> with the endangered <Emphasis Type="Italic">Fagus grandifolia</Emphasis> var. <Emphasis Type="Italic">mexicana</Emphasis>

Gut bacterium Pantoea sp. is one of the predominant bacterial species in the larval gut of the diamondback moth, Plutella xylostella. The phenotypic characters of Pantoea sp. were investigated with BIOLOG phenotype MicroArray (PM) in this study. Totally 950 different metabolic phenotypes were tested using the PM plates 1–10. Results exhibited that Pantoea sp. was able to metabolize 37.37 % of the tested carbon sources, 91.32 % of nitrogen sources, 100 % of sulfur sources, and 98.31 % of phosphorus sources. Most informative utilization patterns for carbon sources of Pantoea sp. were organic acids and carbohydrates, and for nitrogen were various amino acids. The bacterium had 94 different biosynthetic pathways. It had a wide range of adaptabilities, and could still metabolize in osmolytes with up to 9 % sodium chloride, 6 % potassium chloride, 5 % sodium sulfate, 20 % ethylene glycol, 4 % sodium formate, 4 % urea, 5 % sodium lactate, 200 mmol/L sodium phosphate (pH 7.0), 100 mmol/L ammonium sulfate (pH 8.0), 100 mmol/L sodium nitrate, and 100 mmol/L sodium nitrite, respectively. It also exhibited active metabolism under pH values between 4.5 and 10. Pantoea sp. showed active decarboxylase activities while poor deaminase activities in the presence of various amino acids. The phenotypic characterization of Pantoea sp. increased our knowledge of the bacterium, in particular its interactions with insect hosts and the adaptability in gut environments, and showed us some possible approaches to controlling diamondback moth through decreasing Pantoea sp. density.     

Deciphering the mode of action,structural and biochemical analysis of heparinase II/III (<Emphasis Type="Italic">Ps</Emphasis>PL12a) a new member of family 12 polysaccharide lyase from <Emphasis Type="Italic">Pseudopedobacter saltans</Emphasis>

Heparinases are widely used for production of clinically and therapeutically important bioactive oligosaccharides and in analyzing the polydisperse, heterogeneous, and complex structures of heparin/heparan sulfate. In the present study, the gene (1911 bp) encoding heparinase II/III of family 12 polysaccharide lyase (PsPL12a) from Pseudopedobacter saltans was cloned, expressed, and biochemically and functionally characterized. The purified enzyme PsPL12a of molecular size approximately 76 kDa exhibited maximum activity in the temperature range 45–50 °C and at pH 6.0. PsPL12a gave maximum activity at 1% (w/v) heparin under optimum conditions. The kinetic parameters, K _m and V_max, for PsPL12a were 4.6?±?0.5 mg/ml and 70?±?2 U/mg, respectively. Ten millimolars of each Mg²⁺ and Mn²⁺ ions enhanced PsPL12a activity by 80%, whereas Ni²⁺ inhibited by 75% and Co²⁺ by 10%, and EDTA completely inactivated the enzyme. Protein melting curve of PsPL12a gave a single peak at 55 °C and 10 mM Mg²⁺ ions and shifted the peak to 60 °C. The secondary structure analysis of PsPL12a by CD showed 65.12% α-helix, 11.84% β-strand, and 23.04% random coil. The degradation products of heparin by PsPL12a analyzed by ESI-MS spectra displayed peaks corresponding to heparin di-, tetra-, penta-, and hexa-saccharides revealing the endolytic mode of enzyme action. Heparinase II/III (PsPL12a) from P. saltans can be used for production of low molecular weight heparin oligosaccharides for their utilization as anticoagulants. This is the first report on heparinase cloned from P. saltans.     

Screening and identification of an <Emphasis Type="Italic">Enterobacter ludwigii</Emphasis> strain expressing an active <Emphasis Type="Italic">β</Emphasis>-xylosidase

Researchers have expressed increasing interest in the xylanolytic enzymes used in hemicellulose hydrolysis that convert wood and agricultural residues to second-generation biofuels. In our study, 32 isolates showed clear hydrolysis zones on agar plates containing xylan after Congo red staining. Among these isolates, strain LY-62 exhibited the highest β-xylosidase activity (1.29?±?0.05 U/mL). According to the phylogenetic analysis of the 16S rDNA, strain LY-62 belongs to the Enterobacter genus. Using a combination of electron microscopy, Gram-staining, and conventional physiological and biochemical examinations, the strain LY-62 was identified as Enterobacter ludwigii. The β-xylosidase gene from Enterobacter ludwigii LY-62 was cloned, and the full-length protein was expressed in Escherichia coli as an N-terminal or C-terminal His-tagged fusions protein. Optimal β-xylosidase activity was achieved at pH 7.0 and 40 °C. The Michaelis constant K_M values for His-Xyl62 and Xyl62-His were 1.55 and 2.8 mmol/L, respectively. The k_cat values for His-Xyl62 and Xyl62-His were 8.51 and 6.94 s^?1, respectively. The catalytic efficiencies of His-Xyl62 and Xyl62-His were 5.49 and 2.48 s^?1?×?mM^?1, respectively. Thus, Xyl62 is a functional β-xylosidase, and our study represents the first report of a β-xylosidase from Enterobacter ludwigii.     

A novel surfactant-, NaCl-, and protease-tolerant β-mannanase from <Emphasis Type="Italic">Bacillus</Emphasis> sp. HJ14

A glycoside hydrolase family 5 β-mannanase-encoding gene was cloned from Bacillus sp. HJ14 isolated from saline soil in Heijing town. Coding sequence of mature protein (without the predicted signal peptide from M1 to A30) was successfully expressed in Escherichia coli BL21 (DE3). Purified recombinant mannanase (rMan5HJ14) exhibited optimal activity at pH 6.5 and 65 °C. The enzyme showed good salt tolerance, retaining more than 56 % β-mannanase activity at 3.0–30.0 % (w/v) NaCl and more than 94 % of the initial activity after incubation with 3.0–30.0 % (w/v) NaCl at 37 °C for 60 min. Almost no mannanase activity was lost after incubation of rMan5HJ14 with trypsin, proteinase K, and Alcalase at 37 °C for 60 min. Surfactants and chelating agents, namely SDS, CTAB, Tween 80, Triton X-100, EDTA, and sodium tripolyphosphate, showed little or no effect (retaining >82.4 % activity) on enzymatic activity. Liquid detergents, namely Tupperware, Walch, Bluemoon, Tide, and OMO, also showed little or no effect (retaining >72.4 % activity) on enzymatic activity at 0.5–2.0 % (v/v). The enzyme further presents a high proportion (11.97 %) of acidic amino acid residues (D and E), which may affect the SDS and NaCl tolerance of the enzyme. Together, the mannanase may be an alternative for potential use in liquid detergent industry.     

Synthesis of theanine from glutamic acid <Emphasis Type="Italic">γ</Emphasis>-methyl ester and ethylamine catalyzed by <Emphasis Type="Italic">Escherichia coli</Emphasis> having <Emphasis Type="Italic">γ</Emphasis>-glutamyltranspeptidase activity

Glutamic acid γ-methyl ester (GAME) was used as substrate for theanine synthesis catalyzed by Escherichia coli cells possessing γ-glutamyltranspeptidase activity. The yield was about 1.2-fold higher than with glutamine as substrate. The reaction was optimal at pH 10 and 45°C, and the optimal substrate ratio of GAME to ethylamine was 1:10 (mol/mol). With GAME at 100 mmol, 95 mmol theanine was obtained after 8 h.     

Stability of Emulsions Using a New Natural Emulsifier: Sugar Beet Extract <Emphasis Type="Italic">(Beta vulgaris L.)</Emphasis>

This study describes the influence of environmental stresses on the stability of emulsions prepared by a natural sugar beet extract (Beta vulgaris L.). The emulsion stabilizing performance was compared to that of Quillaja extract, which is widely used within the food and beverage industry as natural surfactant. We investigated the influence of pH, ionic strength, heating and freeze-thawing on the mean particle size, ζ-potential and microstructure of oil-in-water emulsions (10% w/w oil, 0.75% w/w emulsifier). The emulsions stabilized by the anionic sugar beet extract were stable at pH 5–8 and against thermal treatments up to 60 °C. However, the prepared emulsions were unstable at acidic (pH 2–4) and basic pH conditions (pH 9), at high temperature (>60 °C), and at salt additions (> 0.1 M NaCl / CaCl₂). Moreover, they also phase separated upon freeze-thawing. Our results show that sugar beet extract is capable of stabilizing emulsions and may therefore be suitable as natural emulsifier for selected applications in the food and beverage industry.     

A physiological comparative study of acid tolerance of <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> ZDY 2013 and <Emphasis Type="Italic">L. plantarum</Emphasis> ATCC 8014 at membrane and cytoplasm levels

This study aimed to disclose the acid tolerance mechanism of Lactobacillus plantarum by comparing L. plantarum ZDY 2013 with the type strain L. plantarum ATCC 8014 in terms of cell membrane, energy metabolism, and amino acid metabolism. L. plantarum ZDY 2013 had a superior growth performance under acidic condition with 100-fold higher survival rate than that of L. plantarum ATCC 8014 at pH 2.5. To determine the acid tolerance physiological mechanism, cell integrity was investigated through scanning electron microscopy. The study revealed that L. plantarum ZDY 2013 maintained cell morphology and integrity, which is much better than L. plantarum ATCC 8014 under acid stress. Analysis of energy metabolism showed that, at pH 5.0, L. plantarum ZDY 2013 enhanced the activity of Na⁺/K⁺-ATPase and decreased the ratio of NAD⁺/NADH in comparison with L. plantarum ATCC 8014. Similarly, amino acid metabolism of intracellular arginine, glutamate, and alanine was improved in L. plantarum ZDY 2013. Correspondingly, the activity of arginine deiminase and glutamate decarboxylase of L. plantarum ZDY 2013 increased by 1.2-fold and 1.3-fold compared with L. plantarum ATCC 8014 in acid stress. In summary, it is demonstrated that the special physiological behaviors (integrity of cell membrane, enhanced energy metabolism, increased amino acid and enzyme level) of L. plantarum ZDY 2013 can protect the cells from acid stress.     

Effects of Formulation Variables on the Particle Size and Drug Encapsulation of Imatinib-Loaded Solid Lipid Nanoparticles

Imatinib (IMT), an anticancer agent, inhibits receptor tyrosine kinases and is characterized by poor aqueous solubility, extensive first-pass metabolism, and rapid clearance. The aims of the current study are to prepare imatinib-loaded solid lipid nanoparticles (IMT-SLN) and study the effects of associated formulation variables on particle size and drug encapsulation on IMT-SLN using an experimental design. IMT-SLN was optimized by use of a “combo” approach involving Plackett-Burman design (PBD) and Box-Behnken design (BBD). PBD screening resulted in the determination of organic-to-aqueous phase ratio (O/A), drug-to-lipid ratio (D/L), and amount of Tween® 20 (Tw20) as three significant variables for particle size (S _z), drug loading (DL), and encapsulation efficiency (EE) of IMT-SLN, which were used for optimization by BBD, yielding an optimized criteria of O/A?=?0.04, D/L?=?0.03, and Tw20?=?2.50%?w/v. The optimized IMT-SLN exhibited monodispersed particles with a size range of 69.0?±?0.9 nm, ζ-potential of ?24.2?±?1.2 mV, and DL and EE of 2.9?±?0.1 and 97.6?±?0.1%?w/w, respectively. Results of in vitro release study showed a sustained release pattern, presumably by diffusion and erosion, with a higher release rate at pH 5.0, compared to pH 7.4. In conclusion, use of the combo experimental design approach enabled clear understanding of the effects of various formulation variables on IMT-SLN and aided in the preparation of a system which exhibited desirable physicochemical and release characteristics.