The Fermentation And Primary Study On Biosynthesizing Of Carotenoids In PSB-W

Carotenoids are pure natural pigment components and have good value in food,cosmetics and medical industries.Chemical synthesis and methods of extracting carotenoids from plants have limitations,and the research on the use of microbial fermentation of carotenoids has broad prospects.A variety of factors have an important impact on the production and extraction of carotenoids.At present,the study of the biosynthesis pathway of the pigment can reveal the process of carotenoid metabolism and achieve the goal of low consumption and high yield.In this paper,a carotenoid-producing strain was screened from aqua purifier.After isolated and purificated the strain,the optimal culture conditions and extraction conditions of high-yield carotenoids were studied.Different conditions were detected by metabolomics.The effects of bacterial fermentation products are intended to provide a basis for the study of carotenoid biosynthesis mechanisms and the development and utilization of photosynthetic bacteria products.The results obtained in this paper are:1.After enriching and cultivating the bacteria in the aqua-purifying agent,a carotenoid-producing bacterium was isolated and purified by four-zone slab scribing.The morphological characteristics of the individual were observed by Gram staining,and the solid-liquid medium was observed.The characteristics of the colony showed that PSB-W is G^-bacteria,which is dark red and viscous in liquid fermentation medium.It is a red round colony on solid medium,combined with physiological and biochemical reactions and 16S rDNA sequence.After the assay,the strain was obtained from the genus Proteiniphilum of the genus Pseudomonas,and the similarity with Proteiniphilum acetatigenes was 99%.2.The culture temperature,light intensity and inoculum size of five different gradients were set up for single factor experiments.The design level of Box-Behnken Design was obtained.The culture conditions of fermented carotenoids were optimized according to BBD design,and the temperature was 29.1℃.The light intensity is 1643 lux,and the amount of carotenoids produced by PSB-W increases when the inoculum is 9.43%.The carotenoid content can reach 8.24 mg/L.3.Five gradients of sodium acetate were added to the medium,and the amount of pigment was measured after anaerobic fermentation for 4 days.The results showed that the amount of carotenoids was significantly increased when sodium acetate was added at 4 g/L.Four kinds of iron salt solutions were prepared and added to the culture medium for fermentation.The OD value of the fermentation broth was sampled every other day.Five concentration gradients were set for each iron salt,and the fermentation broth was added to the fermentation broth for 4 days after anaerobic illumination.Carotenoids,the results show that trivalent iron salts can accelerate the growth of bacteria more than ferrous iron.Appropriate concentration can increase carotenoid content When the concentration of FeCl₃·6H₂O is 300μmol/L,the carotenoids increase significantly.When the concentration of NH₄Fe（SO₄）₂·12H₂O is 400μmol/L,the amount of pigment increases.The concentration of FeSO₄·7H₂O is 200μmol/Carotenoids were significantly increased,and carotenoids were significantly increased at a concentration of（NH₄）₂Fe（SO₄）₂·6H₂O of 400μmol/L.4.The three ways of extracting carotenoids:grinding method,acid heat method,ultrasonic extraction of pigment yield,the amount of carotenoid obtained by ultrasonic extraction is significantly higher than other methods;five different gradients are set.Ultrasonic extraction time,ultrasonic extraction power and ratio of material to liquid were used for single factor experiments.The design level of Box-Behnken Design was obtained.The extraction of carotenoids was optimized according to BBD design.The results were as ultrasonic power was 179.9 W and ultrasonic time was 5.17 min.When the ratio of material to liquid is 0.8,the carotenoid content is increased to 8.6 mg/L.5.The fermentation broth was cultured for 48 h,60 h,72 h and 96 h after the fermentation time was set.The metabolites of each sample at different culture time were detected by metabonomics method.Principal component analysis was used.The differences between the samples were analyzed.The results showed that the samples were significantly different between different fermentation time treatments.The synthesis of amino acids and other metabolites was phased in time,which was related to the growth stage of bacteria.Two kinds of light intensity were set 1000 Lux,1500.The fermentation broth was obtained under Lux,and the metabolites of each sample with different light intensities were detected by metabolomics method.Principal component analysis and orthogonal partial least squares discriminant analysis（OPLS-DA）were used to screen out differential metabolites,combined with KEGG.The pathway database initially obtained the related metabolites in the process of biosynthesis of carotenoids.As a result,bacteria formed acetyl-CoA through pentose phosphate pathway,fatty acid metabolism and metabolism of glycerophosphatidic acid,and acetyl-CoA formed isopentenyl pyrophosphate.Direct substrate,different light intensity has a greater impact on the synthesis of acetyl-CoA,which in turn affects the synthesis of carotenoids.