| β-Carotene is the precursor of vitamin A, the main source of human body’s vitamin A uptake and also an excellent colorant and antioxidant in food industry. Lycopene has good biological functions in antioxidant, anticancer, prevention of cardiovascular disease. β-Carotene and lycopene are two kinds of carotenoids widely used in the fields of functional food, medicine, and cosmetics. The market demand of β-carotene and lycopene were increasing year by year with good commercial prospects. The chemosynthetic lycopene and β-carotene contain a number of byproducts, and have high potential risk of toxicity. The market demand of natural β-carotene and lycopene from the plant tissue extraction and microbial fermentation are growing day by day. Due to the limitations of raw materials, the plant tissue extraction can not meet the needs of large scale and continuous production of carotenoids. Blakeslea trispora (B. trispora) has high content of β-carotene and the carotenoid composition is simple. The strain almost exclusively synthesise three carotenoids (lycopene, y-carotene and β-carotene) and is the only strain that can be used for the industrial fermentation production of natural P-carotene and lycopene.In this study, producing high-quality β-carotene and lycopene by B. trispora was the main research task. Firstly, a simple and rapid analytical method was established to detect the cis and trans isomers of carotenoid; Secondly, a high-efficient screening technique for higher yield of β-carotene by B. trispora was provided; Thirdly, the response mechanism of B. trispora under oxidative stress and the molecular mechanism of oxidative stress on the increasing of β-carotene biosynthesis were studied; Fourthly, a method for producing high-quality lycopene was provided, the lycopene cyclase inhibitors of food and environmental safety were analyzed, and the evaluation criteria of lycopene cyclase inhibitors was established; Fifthly, a clean and efficient extraction method of carotenoid was established.(1) A rapid saponification method is used to completely remove the lipids from the carotenoids for eliminating the interference on detection by using5M potassium hydroxide ethanol solution. The saponated carotenoids can easily dissolve in acetonitrile. A simple, rapid and apolar solvents-free, reverse phase high performance liquid chromatography (HPLC) method is developed for the determination of cis-trans carotenoid isomers. The separation is performed on an Ascentis RP-Amide (15cm×2.1mm,5μm) at room temperature. The mobile phase (acetonitrile) is pumped at a flow rate of0.4mL min1with detection at470nm.(2) In this study, the single colony of B. trispora (+) and B. trispora (-) were obtained by adding0.07%and0.08%of sodium deoxycholate in PDA medium, respectively. Then, the single colony of B. trispora was dyed by0.4%(m/v) triphenyltetrazolium chloride solution (pH7.5). The redder the colony was, the stronger the cell viability was, and greater the potential for high yield of β-carotene was. This method is simple, rapid, and is an effective method for high-efficient screening of B. trispora with high-yield of β-carotene.(3) The cellular response of B. trispora to oxidative stress induced by hydrogen peroxide in shake flask culture was investigated in this study. A mild oxidative stress was created by adding40μM of hydrogen peroxide to a3-day mycelium. The production of β-carotene increased nearly38%after a6-day culture. Under the oxidative stress induced by hydrogen peroxide, the expressions of hmgr, ipi, carG, carRA, and carB involving the β-carotene biosynthetic pathway all increased in3hours. The aerobic metabolism of glucose remarkably accelerated within24hours. In addition, the specific activities of superoxide dismutase and catalase were significantly increased. These changes of B. trispora were responses for reducing cell injury, and the reasons for increasing β-carotene production caused by hydrogen peroxide.(4) This study provided a novel method of producing high-quality lycopene from B. trispora through submerged fermentation.2-Isopropylimidazole at300mg/L completely inhibited lycopene cyclase, which limited the accumulation of γ-carotene and β-carotene.2-Isopropylimidazole also intensified the metabolic flow of lycopene by increasing the expression levels of key enzymes involved in the lycopene synthetic pathway. Ketoconazole at20mg/L reduced the metabolic flow of ergosterol and increased lycopene content and production by76.6%and64.3%, respectively. Lycopene accounted for approximately96%of the total carotenoids, whereas cis-lycopene accounted for approximately35%of the total lycopene. The content and nutritional quality of lycopene from B. trispora were higher than those of lycopene from tomatoes. B. trispora is an ideal source of natural lycopene.(5) This study provided a rapid and environmentally friendly method of carotenoid extraction from B. trispora using steam explosion for cell disruption and ethyl lactate for extraction. Response surface methodology determined the optimal extraction conditions to obtain maximum carotenoid yield. These optimal conditions were as follows:steam explosion at0.20MPa for4.4min, then stirring extraction at a ratio of1:25at39℃for45min using ethyl lactate. By using optimal technology, the yield of total carotenoids reached95.6%. Compared with the conventional extraction method, the time-consuming drying process eliminated, the extraction time is greatly shortened, ethyl lactate was an environmentally friendly and low toxic solvent, and the yield of total carotenoids improved by about31.7%. |
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