A moderate-temperature mutant strain of the yeast Phaffia rhodozyma, termed MK19, was selected by 1-methyl-3-nitro-1-nitrosoguanidine (NTG) and Co60 mutagenesis. MK19 displayed fast cell growth and elevated astaxanthin content at 25 degrees C, whereas optimal temperature for growth and astaxanthin synthesis of wild-type P. rhodozyma was 17-21 degrees C. Optimized astaxanthin yield for MK19 after 4 days culture in shaking flask at 25 degrees C, determined by response surface methodology, was 25.8 mg/l, which was 17-fold higher than that of the wild-type. MK19 was tolerant of high initial concentration of glucose (>100 g/l) in optimized medium. Total fatty acid content of MK19 was much lower than that of the wild-type. Acetyl-CoA is a common precursor of fatty acid and terpenoid biosynthesis, and it is possible that decreased fatty acid synthesis results in transfer of acetyl-CoA to the carotenoid biosynthetic pathway. Our results indicate that astaxanthin content is negatively correlated with fatty acid content in P. rhodozyma. Nutrient analysis showed that MK19 cells are enriched in lysine, vitamin E, and other rare nutrients, and have potential application as fish food without nutritional supplementation. This moderate-temperature mutant strain is a promising candidate for economical industrial-scale production.

Five of 8 fungal lipases screened were found to effectively hydrolyze astaxanthin esters from Haematococcus pluvialis algal cell extracts. Among these, an alkaline lipase from Penicillium cyclopium, expressed in Pichia pastoris, had the highest enzymolysis efficiency. Tween80 was shown to be an effective emulsifier in this lipase hydrolysis system for the 1st time. A series of experiments were performed to find optimal conditions for hydrolysis (pH, temperature, reaction time, lipase dosage). In the optimal reaction system, Tween80 and H. pluvialis extracts (mass ratio 1:1) were emulsified and added to the above lipase at a dosage of 4.6 U/μg (relative to total carotenoids), in phosphate buffer (0.1 M, pH 7.0), and incubated at 28 °C for 7 h, with agitation at 180 rpm. The free astaxanthin recovery ratio under these conditions was 63.2%.

An efficient supercritical fluid extraction (SFE) process with carbon dioxide (SFE-CO(2)) was developed for the extraction of natural vitamin E (V(E)) from wheat germ. Both the pretreatment of extracted wheat germ and extraction conditions were optimized to ensure maximal V(E) yield. The extraction was undertaken at the extracting pressure of 4000-5000 psi, the extracting temperature of 40-45 degrees C, and the carbon dioxide flow rate of 2.0 mL/min for 90 min. An optimized pretreatment of wheat germ was usually necessary with a particle size of 30 mesh and a moisture content of 5.1%. A yield comparison of V(E) and its isomers extracted by supercritical CO(2) with those by conventional solvent extraction suggested that this SFE process was a practical process prospectively superior to conventional solvent extraction to prepare V(E) from wheat germ.

SummaryBoth culture-dependent method and molecular technique were firstly used to simultaneously investigate the cultivable bacterial diversity and amylase production in three typical Daqus of Chinese spirits. The results showed that both cultivable bacterial diversity and amylase production were obviously different. The species of nine bacteria from Deshan, nine from Baisha and six from Wuling Daqus were identified. The total bacterial strains of 17, 15 and 14, and 9, 16 and 10 could produce a-amylase and glucoamylase, respectively, from the Daqus, and the enzyme yields were different. Bacillus licheniformis, Bacillus subtilis and Bacillus amyloliquefaciens not only were dominant bacteria in the Daqus, but also possessed high activities of a-amylase and glucoamylase. By comparison, Bacillus cereus and Bacillus oleronius were found to be another predominant bacterial species and good producers of a-amylase and glucoamylase in Deshan and Wuling Daqus, respectively.

Culture-dependent methods and molecular techniques were used to simultaneously investigate the cultivable bacterial diversity and amylase production in two typical light- lus amyloliquefaciens). All of the bacterial isolates from the Hongxing Daqu could produce extracellular α-amylase with a maximum yield of 25.3 U/ml by B. cereus H17, whereas B. licheniformis H55 could produce a maximum glucoamylase yield of 41.6 U/ml. Some of the bacterial isolates from the Niulanshan Daqu could also produce extracellular α-amylase and glucoamylase. The maximum yield of 27.6 U/ml α-amylase was achieved by B. subtilis N3, and the maximum yield of 58.1 U/ml glucoamylase was achieved by B. cereus N25. Bacillus licheniformis, B. subtilis and B. cereus were not only the dominant bacteria, but also possessed high α-amylase and glucoamylase activities, which may play very important roles during fermentation.