Study On Astaxanthin Production And Regularization Of Metabolism By Xanthophyllomyces Dendrorhous

Astaxanthin(3,3'-dihydroxy-β,β'-carotene-4,4'-dione) is an interesting carotenoid which is the main carotenoid pigment found in aquatic animals such as lobsters, crab, shrimp, trout and salmon and plays a role in delaying or preventing degenerative diseases. Moreover, due to its special structure, astaxanthin is a more powerful scavenger of singlet oxygen (1O2) and peroxyl radicals (H2O2) thanβ-carotene, cantaxanthin, and zeaxanthin (3,3'-dihydroxyl-β-carotene). Its antioxidant activity is much stronger than all other carotenoids. Furthermore, astaxanthin may exert antitumor activities through the enhancement of immune responses. However, consumer and governmental concerns regarding chemical additives in foods have stimulated research in biological systems to produce astaxanthin by biotechnology. These systems are mainly focused on algae, bacteria or yeast such as Xanthophyllomyces dendrorhous. Using different physical and chemical methods, a mutant was isolated and its biological characteristics and production processes of carotenoids were studied. Fast method to determine the astaxanthin and scale-up extract of carotenoids were established, then the parameters of different scale-up fermentation were studied. Based on the astaxanthin biosynthesis pathway by X. dendrorhous, effects of different chemicals and fungal elicitors added into the broth on the growth, total carotenoids and astaxanthin were investigated.To isolate the astaxanthin-hyperproducing yeast, the methods such as different low-dosages of 60Co gamma irradiation, UV and Ethyl Methanesulfonate(EMS) were used to treat X. dendrorhous during exponential phase. Effects of inoculum age, inoculum size, agitation speeds, temperature and initial pH value on the growth, total carotenoids and astaxanthin by X. dendrorhous with optimal conditions of 48 h, 9%, 250 r min–1, 16¹8℃and pH 5.0, respectively. After main nutrition compositions including carbon source, nitrogen source and minerals on carotenoids by X. dendrorhous were determined and optimized, the optimal concentrations of media ingredients such as gluose, sucrose, yeast extract, (NH₄)₂SO₄,KH₂PO₄, Na₂HPO₄ and MgSO₄ were 30,30,6,3,2,1 and 5 g l-1, respectively. Using DMSO as direct extract agent of carotenoids and determination on a spectrophotometer at 492 nm resulted in a fast and approximate method to measure astaxanthin yields by X. dendrorhous. Under the optimal conditions of cell wall disruption which the yeast was treated with 50℃,2M,9ml, 4h and neutralized to pH 3.0, the maximal carotenoid extractability (99. 23%) was obtained.The parameters of different scale-up fermentations were studied in 5, 100, 2000 and 5000 L fermentors, respectively. Influences of operation parameters including agitation speed and aeration were mainly studied in a 5 L fermentor. The results showed that the agitation speed and aeration could significantly affect the growth, total carotenoids, astaxanthin formation and dissolved oxygen by X. dendrorhous. The optimal results were obtained when agitation speed and aeration were 300 r min-1 and 1.5 vvm, respectively. When adding mass water by less times and using dimethyl silicone oil as oxygen vector at concentration of 2% (v/v), the optimal results were obtained by enhancing growth, total carotenoids, astaxanthin formation and dissolved oxygen by X. dendrorhous. 15000 L scale-up experiments which hadn't been reported by any other researcher in this filed was performed in a pharmacy plant. The results of scale-up experiments showed the bigger the fermentor was the shorter the culvitation time was and the dissolved oxygen was a key factor to astaxanthin biosynthesis by X. dendrorhous.Based on astaxanthin biosynthesis by X. dendrorhous, some intermediates such as tomato juice, carrot juice, tri-sodium citrate, ethanol and glutamine added into the culture media could enhance the growth, total carotenoids and astaxanthin formation at different concentrations. Six fungal elicitors prepared from Rhodotorula rubra, Rhodotorula glutinis, Panus conchatus, Coriolus versicolor, Mucor mucedo, Mortieralla alpina stimulate the growth, total carotenoids and astaxanthin formation by X. dendrorhous. Chemical elicitors prepared from TiO₂ and CdCl2 were advantage for total carotenoids and astaxanthin formation but slightly restrained the growth by X. dendrorhous. After being treated with P. conchatus, C. versicolor, TiO₂ and CdCl₂, each elicitor showed a clear positive influence on the reactive oxygen species (ROSs) in the yeast, and there was a steady increase of H2O2 accumulation along with the concentrations, reaching the maximum at certain time at 24 to 40 h with highest H₂O₂ concentration of 299 and 286 nmol g-1 FCW (fresh cell weight) concerning TiO₂ and CdCl₂ elicitor, respectively. The maximal astaxanthin yield was obtained at 96 h after treated by elicitors, which indicated ROSs were due to the sitimulating effects on astaxanthin formation by activating some enzymes and genes involved in astaxanthin biosynthesis pathway by X. dendrorhous.