Optimization Of Biosynthesis Conditions, Structural Characterization And Property Study Of Blue-purple Pigment From Pseudoduganella Sp.QHD

In the thesis,a strain of violet pigment-producing bacteria was isolated and screened from the soil samples,and the structurally characterised of pigment was determined based on the optimization of the synthesis and extraction conditions for pigment production.Subsequently,physicochemical properties and stability of pigment and its biological activities,including antioxidant,antibacterial and antitumour,were investigated.The specific findings of the study are as follows:（1）Isolation,screening and identification of pigment-producing bacteria.The violet pigment-producing bacterium QHDZ isolated and screened from soil samples was identified with morphological,physiological,biochemical,16S r DNA molecular biology assays and phylogenetic tree construction method.The QHDZ strain belongs to Pseudoduganella sp.and was identified to be 99%homologous to Pseudoduganella sp.strain S520.The QHDZ strain is now in storage at the China Center for Type Culture Collection（Collection No.CCTCC NO:M 2021449）.The UV-visible full spectrum scan showed that maximum absorbance of teh pigment is at 570 nm.（2）Optimisation of conditions for the production of violet pigment by strain QHDZ.The combination of single factor and Box-Behnken design-response surface methodology experiments determined the optimization of culture conditions of QHDZ strain.The best culture conditions are as follows:sucrose 3.45%,KNO₃0.09%,K₂PO₄·3H₂O 0.05%,Mg SO₄·7H₂O 0.05%,Na Cl 0.05%,Fe SO₄·7H₂O 0.001%,p H6.05,inoculum size 4%,incubation period 2 d,shaking speed 160 r/min and incubation temperature 25℃.Under these conditions,the pigment potency of strain QHDZ by fermentation was 279.38 U/m L,and the pigment yield was 483.95 mg/m L.（3）Optimization of extraction conditions and structural characterization of the violet pigment QHDZ-P.The combination of single factor and Box-Behnken design-response surface methodology experiments determined violet pigment QHDZ-P extraction conditions:ratio of material to liquid 1:56 g/m L,86%ethanol,extraction temperature40℃,extraction time 60 min,ultrasonic power 200 W.Under these conditions,the violet pigment QHDZ-P extraction could reach 5.16μg/g（wet weight）.TLC（GF254）results showed that the pigment QHDZ-P has two bands on the TLC plate.The Rf value of components I was 0.48,which is lavender colour.The Rf value of components II was 0.4,which is blue colour.The result of column chromatographic separation and purification as follow that petroleum ether-ethyl acetate（V/V=3:1）was used as the unfolding agent.QHDZ-P was isolated on silica gel CC（200-300 mesh）to obtain the lavender fraction QHDZ-P-1 and the blue fraction QHDZ-P-2,its mass ratio is 1:9.The QHDZ-P-2 has a relative molecular mass of 342.13 and molecular formula C₂₀H₁₃O₃N₃using LC-MS、¹H-NMR and ¹³C-NMR analyses,which was identified as violacein.Based on the results of LC-MS analysis,molecular weight differences and literature comparison with QHDZ-P-2,the QHDZ-P-1 has a molecular mass of 326.11 and molecular formula C₂₀H₁₃O₂N₃,which was identified as deoxyviolacein.（4）Studies on the physicochemical properties and stability of the violet pigment QHDZ-P.QHDZ-P is insoluble in water,soluble in ethyl acetate,acetone,anhydrous ethanol and 2%SDS solution,readily soluble in anhydrous ethanol and acetone.Natural light and UV light have a significant effect on the stability of pigment and caused the pigment to fade.Al³⁺and Ca²⁺had no significant effect on the stability of the pigment,while Fe³⁺,Cu²⁺and Zn²⁺had a greater effect on the stability of the violet pigment QHDZ-P.Sucrose,ascorbic acid,citric acid,sodium chloride,potassium sorbate and disodium hydrogen phosphate had an effect on the stability of the colour.The oxidizing agent KMn SO₄had a strong influence on the stability of the violet pigment QHDZ-P.The reducing agent Fe SO₄has a certain colour enhancing effect on the pigment.The pigment is thermally stable and has good stability in the p H range 2-12.Therefore,the pigment should be stored away from light and oxygen and should not come into contact with Mg²⁺,Fe³⁺,Cu²⁺and Zn²⁺.（5）In vitro antioxidant and antibacterial properties of the violet pigment QHDZ-P.Antioxidant activity of violet pigment QHDZ-P against DPPH radicals,hydroxyl radicals and ABTS radicals was determined by DPPH assay,salicylic acid assay and ABTS assay,and results show that the pigment QHDZ-P has the ability to scavenge both DPPH and hydroxyl radicals at concentrations ranging from 0.01 mg/m L to 0.05 mg/m L,and showed a positive dose-effect correlation,its scavenging of DPPH radicals was higher than that of the same concentration of vitamin E,while its scavenging of hydroxyl radicals was lower than that of the same concentration of vitamin E.The pigment QHDZ-P concentrations in the range of 0.05 mg/m L-0.25 mg/m L showed high scavenging capacity for ABTS radicals,and at 0.25 mg/m L,its scavenging rate was approximately three times higher than vitamin E with the same concentration.The results of the perforation method to determine the antibacterial activity of the violet pigment QHDZ-P showed that the pigment QHDZ-P concentrations in the range of 3.125 mg/m L to 50 mg/m L did not inhibit the growth of E.coli and S.aureus.（6）In vitro antitumor activity study of the violet pigment QHDZ-P-2.Non-small cell lung cancer A549 cells and human degenerative cancer Calu-6 cells were cultured in vitro,and the pigment QHDZ-P-2 was administered by microscopic observation,the results revealed that the original morphology of tumour cells was significantly inhibited after QHDZ-P-2 incubation,with the reduction of cell number and nuclear consolidation.The results of the MTT experiment showed that the pigment QHDZ-P-2 had a proliferation inhibitory effect on non-smoll cell lung cancer A549 cells,the inhibitory effect was positively correlated with the administered concentration between 6.25μmol/m L and 100μmol/m L.The pigment QHDZ-P-2 significantly induced apoptosis in lung cancer A549 cells,concentrations in the range of 50μmol/m L-200μmol/m L,the apoptosis rate increased with increasing dosing concentration.The non-smoll cell lung cancer A549 apoptotic cells which incubated with the violet QHDZ-P-2 caused insignificant DNA band breaks.It indicated that apoptosis occurs at a late stage and is positively correlated with the concentration of drug administered.