Preparation and characterization of astaxanthin-loaded biodegradable polyhydroxybutyrate (PHB) microbeads for personal care and cosmetic applications.

Due to its biodegradability and biocompatibility, polyhydroxybutyrate (PHB) has received attention as an alternative material for microbeads in personal care and cosmetic products (PCCPs). Here, PHB was produced from crude glycerol by an Escherichia coli JM109 strain harboring pUC19-23,119-phaCABA-04 without isopropyl ß-D-1-thiogalactopyranoside (IPTG), an inducing agent. Astaxanthin-loaded PHB microbeads were prepared through emulsification-solvent evaporation. Studies were performed to determine how the concentration of PHB and stirring rate influence the size, surface morphology, encapsulation efficiency (EE), and astaxanthin release profile. The astaxanthin-loaded PHB microbeads exhibited a rough surface, 98.1 ± 0.7 % EE, spherical shape and 179 ± 44 µm size. In addition, <50 % astaxanthin release was observed within 240 min. Stability studies revealed that astaxanthin-loaded microbeads retained over 85.3 ± 4.2 % of astaxanthin after 90 days at 4 °C and showed a 2-fold reduction in astaxanthin degradation compared to their unencapsulated counterparts; thus, astaxanthin-loaded microbeads show promise for PCCPs applications. A cytotoxicity assay revealed that astaxanthin-loaded PHB microbeads were nontoxic to the human epidermal keratinocyte cell line, PSVK1, and EpiSkin® cells. Skin irritation and sensitization were not observed during a human repeated insult patch test (HRIPT), according to clinical practice guidelines of the Japanese dermatological association.

Strategies for Poly(3-hydroxybutyrate) Production Using a Cold-Shock Promoter in Escherichia coli.

The present study attempted to increase poly(3-hydroxybutyrate) (PHB) production by improving expression of PHB biosynthesis operon derived from Cupriavidus necator strain A-04 using various types of promoters. The intact PHB biosynthesis operon of C. necator A-04, an alkaline tolerant strain isolated in Thailand with a high degree of 16S rRNA sequence similarity with C. necator H16, was subcloned into pGEX-6P-1, pColdI, pColdTF, pBAD/Thio-TOPO, and pUC19 (native promoter) and transformed into Escherichia coli JM109. While the phaC A-04 gene was insoluble in most expression systems tested, it became soluble when it was expressed as a fusion protein with trigger factor (TF), a ribosome associated bacterial chaperone, under the control of a cold shock promoter. Careful optimization indicates that the cold-shock cspA promoter enhanced phaCA-04 protein expression and the chaperone function of TF play critical roles in increasing soluble phaCA-04 protein. Induction strategies and parameters in flask experiments were optimized to obtain high expression of soluble PhaCA-04 protein with high YP/S and PHB productivity. Soluble phaCA-04 was purified through immobilized metal affinity chromatography (IMAC). The results demonstrated that the soluble phaCA-04 from pColdTF-phaCAB A-04 was expressed at a level of as high as 47.4 ± 2.4% of total protein and pColdTF-phaCAB A-04 enhanced soluble protein formation to approximately 3.09-4.1 times higher than that from pColdI-phaCAB A-04 by both conventional method and short induction method developed in this study. Cultivation in a 5-L fermenter led to PHB production of 89.8 ± 2.3% PHB content, a YP/S value of 0.38 g PHB/g glucose and a productivity of 0.43 g PHB/(L.h) using pColdTF-phaCAB A-04. The PHB film exhibited high optical transparency and possessed Mw 5.79 × 105 Da, Mn 1.86 × 105 Da, and PDI 3.11 with normal melting temperature and mechanical properties.

Medium chain length polyhydroxyalkanoates consisting primarily of unsaturated 3-hydroxy-5-cis-dodecanoate synthesized by newly isolated bacteria using crude glycerol.

BACKGROUND: Our study aimed to search for novel bacteria capable of producing polyhydroxyalkanoates (PHAs) using crude glycerol residue obtained from biodiesel production in which used cooking oils were the substrates. RESULTS: Newly isolated bacteria from soils in Thailand were screened for the efficient production of PHAs from crude glycerol. The bacterial strains were cultivated on glucose, refined glycerol, crude glycerol, or various cooking oils (canola oil, palm oil, soybean oil, sunflower oil, corn oil, grape seed oil, olive oil, rice bran oil, camellia seed oil) for growth and PHA production. The effects of the total organic carbon (TOC) concentration and the mole ratio of carbon to nitrogen were investigated in batch cultivation. (1)H NMR, two dimensional-(1)H-correlation spectroscopy (2D-(1)H-COSY) and (13)C NMR analyses confirmed four bacterial strains were capable of producing medium-chain-length PHAs (mcl-PHAs), consisting of 3-hydroxyoctanoate (3HO) and 3-hydroxy-5-cis-dodecanoate (3H5DD), from crude glycerol. On the basis of phenotypic features and genotypic investigations, the bacterial strains were assigned as: ASC1, Acinetobacter genus (94.9% similarity); ASC2, Pseudomonas genus (99.2% similarity); ASC3, Enterobacter genus (99.2% similarity); ASC4, Bacillus genus (98.4% similarity). The highest amount of mcl-PHAs, 17.5 ± 0.8 g/L (content 61.8 ± 3.3% wt), with 3HO (14.7 ± 2.2 mol %), 3H5DD (85.3 ± 2.2 mol%), and a total biomass of 32.3 ± 0.3 g/L, was obtained from Pseudomonas sp. ASC2 in batch cultivation after 36 h. The mcl-PHAs recovered had a number-average molecular weight (M N) of 3.6 × 10(4) Da. Homopolymeric 3H5DD was obtained when the cultivation time was prolonged to 96 h. CONCLUSIONS: Novel PHA-producing strains were isolated and identified. These bacterial strains are able to produce mcl-PHAs from crude glycerol. The mcl-PHAs produced contained a high percentage of 3H5DD, which suggests their future application as softeners mixed with other biomaterials. The unsaturated side chain of 3H5DD monomers containing double bounds offers additional potential for improving the properties of the mcl-PHAs or extending their applications to the food industry.