Tarek S. Awad

Effect of surfactant surface coverage on formation of solid lipid nanoparticles (SLN)

The effect of surfactant surface coverage on formation and stability of Tween 20 stabilized tripalmitin solid lipid nanoparticles (SLN) was investigated. A lipid phase (10% w/w tripalmitin) and an aqueous phase (2% w/w Tween 20, 10 mM phosphate buffer, pH 7) were heated to 75 degrees C and then homogenized using a microfluidizer. The resulting oil-in-water emulsion was kept at a temperature (37 degrees C) above the crystallization temperature of the tripalmitin to prevent solidification of emulsion droplets, and additional surfactant at various concentrations (0-5% w/w Tween 20) was added. Droplets were then cooled to 5 degrees C to initiate crystallization and stored at 20 degrees C for 24 h. Particle size and/or aggregation were examined visually and by light scattering, and crystallization behavior was examined by differential scanning calorimetry (DSC). Excess Tween 20 concentration remaining in the aqueous phase was measured by surface tensiometry. Emulsion droplets after homogenization had a mean particle diameter of 134.1+/-2.0 nm and a polydispersity index of 0.08+/-0.01. After cooling to 5 degrees C at low Tween 20 concentrations, SLN dispersions rapidly gelled due to aggregation of particles driven by hydrophobic attraction between insufficiently covered lipid crystal surfaces. Upon addition of 1-5% w/w Tween 20, SLN dispersions became increasingly stable. At low added Tween 20 concentration (<1% w/w) the SLN formed gels but only increased slightly at higher surfactant concentrations (>1% w/w). The Tween 20 concentration in the aqueous phase decreased after tripalmitin crystallization suggesting additional surfactant adsorption onto solid surfaces. At higher Tween 20 concentrations, SLN had increasingly complex crystal structures as evidenced by the appearance of additional thermal transition peaks in the DSC. The results suggest that surfactant coverage at the interface may influence crystal structure and stability of solid lipid nanoparticles via surface-mediated crystal growth.

Impact of Surfactant Properties on Oxidative Stability of β-Carotene Encapsulated within Solid Lipid Nanoparticles

The impact of surfactant type on the physical and chemical stability of solid lipid nanoparticle (SLN) suspensions containing encapsulated beta-carotene was investigated. Oil-in-water emulsions were formed by homogenizing 10% w/w lipid phase (1 mg/g beta-carotene in carrier lipid) and 90% w/w aqueous phase (surfactant + cosurfactant) at pH 7 and 75 degrees C and then cooling to 20 degrees C. The impact of surfactant type was investigated using aqueous phases containing different water-soluble surfactants [2.4% w/w high-melting (HM) lecithin, 2.4% w/w low-melting (LM) lecithin, and 1.4% w/w Tween 60 or 1.4% w/w Tween 80] and a cosurfactant (0.6% taurodeoxycholate). The impact of the physical state of the carrier lipid was investigated by using either a high melting point lipid (tripalmitin) to form solid particles or a low melting point lipid (medium chain triglycerides, MCT) to form liquid droplets. A higher fraction of alpha-crystals was detected in solid particles prepared with high-melting surfactants (HM-lecithin and Tween 60) than with low-melting surfactants (LM-lecithin and Tween 80). With the exception of the HM-lecithin-coated solid particles, the suspensions were stable to particle aggregation during 21 days of storage. beta-Carotene degradation after 21 days of storage was 11, 97, 100, and 91% in the solid particles (tripalmitin) and 16, 21, 95, and 90% in the liquid droplets (MCT) for HM-lecithin, LM-lecithin, Tween 80, and Tween 60, respectively. These results suggest that beta-carotene may be stabilized by (1) LM- or HM-lecithin when liquid carrier lipids are used and (2) HM-lecithin when solid carrier lipids are used. The origin of this latter effect is attributed to the impact of the surfactant tails on the generation of a crystal structure better suited to maintain the chemical stability of the encapsulated bioactive.

Deinococcus aquiradiocola sp. nov., isolated from a radioactive site in Japan

A gamma- and UV-radiation-tolerant, pale-pink strain (TDMA-uv53T) was isolated from a freshwater sample collected at Misasa (Tottori, Japan), after exposure of the water sample to UV radiation. The cells stained Gram-positive and were non-motile, rod-shaped and non-spore-forming. The DNA G+C content of the strain was 69.1 mol%. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain TDMA-uv53T belongs to the genus Deinococcus, the highest sequence similarities being found with Deinococcus claudionis PO-04-19-125T (96 %), D. altitudinis ME-04-01-32T (96 %), D. radiomollis PO-04-20-132T (95 %), D. deserti VCD115T (91.5 %), D. hopiensis KR-140T (91.0 %) and D. sonorensis KR-87T (91.0 %). Major fatty acids were iso-15 : 0, 15 : 1omega6c, 15 : 0, 16 : 0 and summed feature 3 (iso-15 : 0 2-OH and/or 16 : 1omega7c). MK-8 was the predominant respiratory quinone. Phylogenetic distinctiveness and unique phenotypic characteristics differentiated strain TDMA-uv53T from closely related Deinococcus species. The results of our polyphasic taxonomic analyses suggested that TDMA-uv53T represents a novel Deinococcus species, for which the name Deinococcus aquiradiocola sp. nov. is proposed. The type strain is TDMA-uv53T (=JCM 14371(T) [corrected] =NBRC 102118T =CCUG 53612T).

Deinococcus depolymerans sp. nov., a gamma- and UV-radiation-resistant bacterium, isolated from a naturally radioactive site

Four gamma- and UV-radiation-resistant bacterial strains, designated TDMA-24(T), TDMA-24-2, TDMA-24-3 and TDMA-24-4, were isolated from a fresh-water sample collected at Misasa, Tottori, Japan. Cells of these strains were Gram-reaction-positive, non-motile, non-spore-forming, rod-shaped and formed red colonies. The genomic DNA G+C contents ranged from 70.5 to 70.6 mol%. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the novel isolates belong to the genus Deinococcus, the highest sequence similarities being with Deinococcus aquaticus PB314(T) (98%) and Deinococcus caeni Ho-08(T) (97 %). The polar lipid profile of strain TDMA-24(T) comprised three unidentified phosphoglycolipids, five unidentified glycolipids and seven unidentified polar lipids. MK-8 was the predominant respiratory quinone. Major fatty acids were iso-C(15 : 0), C(15 : 1)ω6c, C(15 : 0), C(16 : 0) and summed feature 3 (iso-C(15 : 0) 2-OH and/or C(16 : 1)ω7c). On the basis of their phylogenetic positions and chemotaxonomic and phenotypic characteristics, the novel isolates represent a novel species of the genus Deinococcus, for which the name Deinococcus depolymerans sp. nov. is proposed. The type strain is TDMA-24(T) ( = JCM 14369(T)  = NBRC 102115(T)  = CCUG 53609(T)).

Lipids of Haloferax alexandrinus strain TMT: an extremely halophilic canthaxanthin-producing archaeon

The core lipids, polar and non polar lipids, of the novel canthaxanthin-producing archaeon, Haloferax alexandrinus strain TM(T), were investigated using thin-layer chromatography (TLC), spectrophotometry, electron ionization-mass spectroscopy (EI-MS), fast atom bombardment mass spectrometry (FAB-MS), nuclear magnetic response spectrometry (13C-NMR) and high performance liquid chromatography (HPLC). This archaeon appeared to contain diphytanyl diether lipids (C20-C20) as core lipids. The major phospholipids were found to be phosphatidylglycero-phosphate-methyl ester (PGP-Me) and phosphatidylglycerol (PG), but no phosphatidylglycero-sulfate was detected. The strain contained two glycolipids, sulfated diglycosyl diether (S-DGD-1) and unsulfated diglycosyl diether (DGD). Analysis of the non polar lipids revealed the presence of beta-carotene, 3-hydroxyechinenone, gamma-carotene, cis-astaxanthin, lycopene, trisanhydro-bacterioruberin,monanhydro-bacterioruberin, bacterioruberin isomer, bacterioruberin and canthaxanthin. Although the polar lipids profile of Hfx. alexandrinus strain TM(T) was similar to those of other species of the genus Haloferax, the non polar lipid (carotenoids) profile was markedly different. Further experiments on the influence of NaCl concentration on the lipids composition of Hfx. alexandrinus strain TM(T) demonstrated the dependence of the proportion of each of bacterioruberin, beta-carotene and canthaxanthin on the NaCl concentration in the growth media.

Temperature scanning ultrasonic velocity study of complex thermal transformations in solid lipid nanoparticles

The purpose of this study was to determine whether temperature scanning ultrasonic velocity measurements could be used to monitor the complex thermal transitions that occur during the crystallization and melting of triglyceride solid lipid nanoparticles (SLNs). Ultrasonic velocity ( u) measurements were compared with differential scanning calorimetry (DSC) measurements on tripalmitin emulsions that were cooled (from 75 to 5 degrees C) and then heated (from 5 to 75 degrees C) at 0.3 degrees C min (-1). There was an excellent correspondence between the thermal transitions observed in deltaDelta u/delta T versus temperature curves determined by ultrasound and heat flow versus temperature curves determined by DSC. In particular, both techniques were sensitive to the complex melting behavior of the solidified tripalmitin, which was attributed to the dependence of the melting point of the SLNs on particle size. These studies suggest that temperature scanning ultrasonic velocity measurements may prove to be a useful alternative to conventional DSC techniques for monitoring phase transitions in colloidal systems.

A Novel Radio-Tolerant Astaxanthin-Producing Bacterium Reveals a New Astaxanthin Derivative: Astaxanthin Dirhamnoside

Astaxanthin is a red ketocarotenoid that exhibits extraordinary health-promoting activities such as antioxidant, anti-inflammatory, antitumor, and immune booster. The recent discovery of the beneficial roles of astaxanthin against many degenerative diseases such as cancers, heart diseases, and exercise-induced fatigue has raised its market demand as a nutraceutical and medicinal ingredient in aquaculture, food, and pharmaceutical industries. To satisfy the growing demand for this high-value nutraceuticals ingredient and consumer interest in natural products, many research efforts are being made to discover novel microbial producers with effective biotechnological production of astaxanthin. Using a rapid screening method based on 16S rRNA gene, and effective HPLC-Diodearray-MS methods for carotenoids analysis, we succeeded to isolate a unique astaxanthin-producing bacterium (strain TDMA-17(T)) that belongs to the family Sphingomonadaceae (Asker et al., Appl Microbiol Biotechnol 77: 383-392, 2007). In this chapter, we provide a detailed description of effective HPLC-Diodearray-MS methods for rapid analysis and identification of the carotenoids produced by strain TDMA-17(T). We also describe the methods of isolation and identification for a novel bacterial carotenoid (astaxanthin derivative), a major carotenoid that is produced by strain TDMA-17(T). Finally, we describe the polyphasic taxonomic analysis of strain TDMA-17(T) and the description of a novel species belonging to genus Sphingomonas.

Screening and profiling of natural ketocarotenoids from environmental aquatic bacterial isolates

Ketocarotenoids are high-value natural pigments. The red diketocarotenoid astaxanthin particularly exhibits an extraordinary antioxidant activity, which raises its market demand for foods and nutraceuticals. We screened for ketocarotenoid-producing bacteria from both marine and freshwater environments. Phylogenetic analysis, based on 16S rRNA gene sequence, revealed 37 potential producers of ketocarotenoids that are related to α-proteobacteria, comprising 32 strains of Brevundimonas and 5 strains of Erythrobacter. Carotenoids analysis by HPLC-DAD and HPLC-MS revealed two groups; astaxanthin-producers (28 Brevundimonas strains) and adonixanthin-producers (Five Brevundimonas and 5 Erythrobacter strains). Strain FrW-Asx16 exhibited the highest carotenoid production (1060 µg g-1 dry cells with 16.6% astaxanthin). Strain FrW-Asx-5 producing 946.1 µg g-1 dry cells carotenoid exhibited the highest astaxanthin content (∼46%). The most intriguing result is the potential of producing natural colorants from freshwater bacterial isolates, and with high productivity and selectivity, suggesting a great promise for their application in food.

Corrigendum: Deinococcus aquiradiocola sp. nov., isolated from a radioactive site in Japan

A third affiliation for author Dalal Asker was not added. The full list of author affiliations is below: Dalal Asker, Tarek S. Awad, Teruhiko Beppu, Kenji Ueda 1 Life Science Research Center, College of Bioresource Sciences, Nihon University, 1866 Kameino, Fujisawa 252-8510, Japan 2 Department of Food Science, University of Massachusetts, 100 Holdsworth Way, Amherst, MA 01003, USA 3 Department of Food Science & Technology, Alexandria University, Alexandria, Egypt

Purification and Identification of Astaxanthin and Its Novel Derivative Produced by Radio-tolerant Sphingomonas astaxanthinifaciens

The red diketocarotenoid, astaxanthin, exhibits extraordinary health-promoting activities such as antioxidant, anti-inflammatory, antitumor, and immune booster, which may potentially protect against many degenerative diseases such as cancers, heart diseases, and exercise-induced fatigue. These numerous health benefits and consumer interest in natural products have therefore increased the market demand of astaxanthin as a nutraceutical and medicinal ingredient in food, aquaculture feed, and pharmaceutical industries. Consequently, many research efforts have been made to discover novel microbial sources with effective biotechnological production of astaxanthin. Using a rapid screening method based on 16S rRNA gene, and effective HPLC-Diode array-MS methods for carotenoids analysis, we isolated a novel astaxanthin-producing bacterium (strain TDMA-17T) that belongs to the family Sphingomonadaceae (Asker et al., FEMS Microbiol Lett 273:140-148, 2007).In this chapter, we provide a comprehensive description of the methods used for the analysis and identification of carotenoids produced by strain TDMA-17T. We will also describe the methods of isolation and identification for a novel bacterial carotenoid (an astaxanthin derivative), a major carotenoid that is produced by the novel strain. Finally, the identification methods of the novel strain will be summarized.