Optimization Of Fermentation,Purification And Activity Of Blue Pigments From Quambalaria Cyanescens QY229

Blue pigment is widely used in food,medicine,cosmetics and other fields.At present,the majority of blue pigments for sale are chemical synthetic blue pigments,which have safety risks.Therefore,the development of new natural blue pigments has attracted attention at home and abroad.In this study,Quambalaria cyanescens blue pigment was reported for the first time.The fermentation medium and culture conditions of blue pigments produced by Quambalaria cyanescens QY229 were optimized,and the separation and purification,physicochemical properties and biological activities of blue pigments were studied.1.Optimization of liquid-state fermentation medium and culture conditions for production of blue pigments from Quambalaria cyanescens QY229.The optimal carbon and nitrogen sources were glucose and peptone by single factor experiment.Based on Plackett-Burman（PB）experiment,three key factors including nitrogen source（peptone）concentration,culture temperature and medium volume were screened from nine factors.The path of steepest ascent method combined with Box-Behnken（BBD）experiment design was used for response surface（RSM）analysis of the selected important factors,and the optimal fermentation parameters for the production of blue pigments by Quambalaria cyanescens QY229 were obtained:Peptone concentration was 34.6 g·L^-1,culture temperature was 31.7℃,medium volume in a 250 m L flask was 72.3 m L.The predicted color value of fermentation broth containing blue pigment was 350.4±6.8 U·m L^-1.The actual color value of fermentation broth was 348.2±7.1 U·m L^-1,which had no significant difference from the predicted value（p>0.05）.The optimized fermentation liquid color value is 2.1times of that before optimization.2.Isolation and purification of blue pigment from Quambalaria cyanescens QY229.First,the supernatant of fermentation of Quambalaria cyanescens QY229 was clarified by ethanol precipitation method.Then,microfiltration（MF）,ultrafiltration（UF）,Sephadex LH-20 gel column chromatography（CC）,preparative high performance liquid chromatography（Prep-HPLC）and other methods were used to separate and purify blue pigment.The purified blue pigment was identified by high performance liquid chromatography（HPLC）,and the final purity was more than 95%.3.Study on solubility,color change,spectral characteristics and other physicochemical properties and stability of QY229 blue pigment from Quambalaria cyanescens.The results showed that blue pigment was blue purple powder after freeze-drying at natural p H,soluble in water,water and methanol or water and ethanol mixture,insoluble in anhydrous methanol,ethanol,acetone,ethyl acetate,acetonitrile,ether,chloroform and petroleum ether and other organic solvents.Blue pigment has strong stability in the acid environment below 60℃and avoid light.Metal ions Zn²⁺and Mg²⁺have obvious color enhancement effect on blue pigment solution,K⁺,Na⁺and Li⁺have no obvious effect on the stability of blue pigment,while Ca²⁺,Fe²⁺,Mn²⁺,Cu²⁺,Al³⁺,Fe³⁺precipitate with pigment.The acid blue pigment solution has tolerance to oxidant（H₂O₂）,and the addition of common food additives（Sodium cyclamate,mannitol,malic acid,citric acid,vitamin C and sodium benzoate）and cosmetic additives（glycerin,butanediol）has no significant effect on the stability of blue pigment.Neutral to alkaline blue pigment solutions were tolerant to reducing agent（Na₂SO₃）.In addition to malic acid,citric acid and vitamin C,the addition of other food and cosmetic additives had little effect on the stability of blue pigment.4.Analysis of antioxidant activity andα-glucosidase inhibitory activity of QY229blue pigment in vitro.The results showed that when the concentration of blue pigment≥150μg·m L^-1,the maximum scavenging rates of DPPH,ABTS and hydroxyl radicals were above 97.00%.At the same concentration(50μg·m L^-1),the reducing capacity of blue pigment and ascorbic acid to Fe³⁺ion was 0.86±0.02 mmol·L^-1and 0.87±0.02mmol·L^-1,respectively,and there was no significant difference between them（p>0.05）.When the concentration of blue pigment≥180μg·m L^-1,the inhibitory activity ofα-glucosidase reached the maximum,and the inhibitory rate was 58.16±2.53%.5.Acute toxicity（24 h）and biological activity analysis of QY229 pigment against C.elegans.The results showed that 0.00～1.25 mg·m L^-1blue pigment was non-toxic to C.elegans.The C.elegans were treated with 0.05,0.25 and 1.25 mg·m L^-1blue pigments,the body length of C.elegans increased by 44.36±2.11%～55.45±3.23%;head swing frequency increased by 8.27±0.81%～19.54±1.32%;body expansion frequency increased 5.41±0.45%～36.57±1.98%;pharyngeal pump rate increased by 4.82±0.32%～36.23±1.45%;the excretion cycle is shortened by 2.20±0.13%～20.08±1.21%;prolong life by 22.22±1.32%～33.33±1.78%;the survival time of oxidative stress was increased by 6.90±0.32%～20.69±1.22%.In the heat stress test,the survival time was increased by 9.09±0.82%～27.27±2.34%,CAT enzyme activity was increased by 22.22±2.04%～39.96±3.33%,SOD enzyme activity was increased by 1.86±0.22～2.68±0.43.The expression levels of genes associated with longevity（daf-16,skn-1,sod-3,ctl-1,hsf-1,hsp-16.2,gcs-1 and gst-4）were significantly upregulated（p<0.05）.Therefore,QY229 blue pigment has anti-aging effect on C.elegans.