Study On Carotenoid Producing Microorganism Isolation,culture Conditions Optimization And Their Pigment

Carotenoids are kinds of terpenoid compounds and their derivatives widely existing in nature.They are mainly found in plants and microbes.They have many physiological functions such as antioxidant,vitamin A,anticancer and resistance to ultraviolet radiation.Carotenoid is widely used in food,medicine and chemical industry because of its bright color and unique physiological function.The use of"the standard of national food safety,standards of food additives"?GB2760-2014?pointed out that natural carotenoids can accord to the needs of production as a food colorant added to all kinds of food in addition to pasteurized milk,cream,fresh fruits and vegetables.Compared with carotenoids from plants,microbial source carotenoids are widely studied due to their short production cycle,unrestricted raw material area and relatively simple extraction process.This paper aims to the separation and identificaty of carotenoid producing microorganisms,exploration the carotenoid extraction and stability,then study carotenoid purification,structure identification and antioxidant activity.The purpose is to meet the needs of food,medicine and cosmetics industry on carotenoid purity,solubility,stability and physiological activity,broaden the the actual application scope.The main contents and conclusions are as follows:1.Screening and identification of carotenoid producing microorganisms.Carotenoid producing strain UL is screened from the Jialing River in Beibei District,Chongqing.After morphological,physiological and biochemical,molecular biological identification and phylogenetic tree construction,it showed that the UL strain was Acinetobacter lwoffii,which was 97%homologous with Acinetobacter lwoffii NCTC5866.UL strain is now preserved in China's typical culture center,the name is Acinetobacter lwoffii UL,and the preservation number is CCTCC M 2017776.2.Preliminary identification pigment of Acinetobacter lwoffii UL and optimization of culture conditions.Pigment-chloroform solution and sulfuric acid in the reaction interface with blue and green color and it was carotenoids.The UV-visible spectrum of the pigment shows that the line has three finger shape and the maximum absorbance value is 478 nm,which accords with the UV visible spectrum characteristics of carotenoids.The optimization of culture conditions of UL strain shows that the best culture conditions of UL strain are 108 h,inoculation volume 0.4%,pH6.5,liquid volume 90 mL,shaking 120 r/min and incubation temperature 30?.3.Optimization of extraction conditions of Acinetobacter lwoffii UL.The combination of single factor and Box-Behnken determine carotenoid pigment extraction of the Acinetobacter lwoffii UL.They are 90%ethanol,extraction temperature 27?,extraction time 30 min,ratio of material to liquid 7:50 g/mL.Influence of various factors on the extraction effect of the order is solid-liquid ratio?C?,extraction temperature?B?,extraction time?A?.The extraction time and temperature,extraction temperature and ratio of material to liquid interaction significantly,B² and C²respectively showed extremely significant and significant,carotenoid pigment can reach5.16?g/g?wet weight?.4.Acinetobacter lwoffii UL carotenoids stability.Testing the effects of light,temperature,pH,oxidant,food additives and metal ions on the stability of carotenoids,results show that carotenoids extract in indoor light,low temperature is stable,but have adverse effects in the presence of sunlight,temperature higher than 55?,sodium benzoate,vitamin C,Cu²⁺,Fe³⁺,Fe²⁺.At the same time,carotenoids have certain antioxidant activity.5.Purification and identification carotenoid of Acinetobacter lwoffii UL.After TLC and column chromatography test,the optimum expansion condition of TLC was n-heptane:acetone=1:1,two red bands were obtained,and R_f was 0.428 and 0.5,respectively.The best elution conditions for column chromatography are flow rate 1mL/min,sample volume 4 mL,eluent n-heptane:acetone=1:1,and 4 min receiver.The system can be divided into two components,components 1 and components 2.In view of a little of component 1,only the components 2 were tested.Solubility,HPLC,1H-NMR,FTIR and UPLC-Q-TOF-MS-MS identification,the results show that the pigment is insoluble in water.Although purified,the pigment still contains similar structure components.On the basis of FRIT,1H-NMR and UPLC-Q-TOF-MS-MS results infer that carotenoid components 2 may contain Astacen,molecular formula C₄₀H₄₈O₄,molecular weight 592;astaxanthin,molecular formula C₄₀H₅₂O₄,molecular weight 596.6.Detection carotenoids antioxidant activity of Acinetobacter lwoffii UL.ABTS,FRAP,DPPH to evaluate the carotenoid crude extract and purified carotenoid component 1 and 2,results showed that purified carotenoid antioxidant activity was higher.By the method of FRAP evaluation,carotenoid composition 1 antioxidant activity is crude extracts 7.23 times,composition 2 antioxidant activity is crude extracts7.79 times;with the DPPH method,composition 1 antioxidant activity is crude extracts5.52 times,carotenoid composition 2 antioxidant activity is crude extracts 5.48 times.