PH-sensitivity Of Monascus Pigments And Metabolic Regulation Of Monascus Pigments In Extractive Fermentation Under Low PH

Monascus pigments are secondary metabolites with polyketide structures of Monascus spp.with a long history of usage as food natural colorants in China and many other East and Southeast Asian countries.Nowadays,more and more people pay attention to the issue of food safety,and the high security and various color characteristics of Monascus pigments make them more competitive as food natural colorants.In this study,the major Monascus pigments were separated and purified from the mycelia of submerged fermentation.Then the pH-sensitivity of these pigments were investigated,including the pH-sensitive spectral properties,color characteristics,reactivity properties and photodegradation properties;the synergistic regulation of pH and nitrogen sources on Monascus pigment metabolism was also investigated;then the consequent impact of these properties on the analysis,application and metabolic regulation of Monascus pigments were further studied and discussed.Also,the pigment metabolism and regulation in extractive fermentation with a pigment high-yield strain(Monascus anka GIM 3.592)under low pH were also investigated and discussed;the differences in pigment fingerprint profiles between submerged and extractive fermentations were investigated;at last,extractive fermentation and immobilized cell fermentation were combined to continuously produce Monascus yellow pigments extracellularly.The major conclusions are as follows:(1)The spectra and color characteristics of orange pigments(rubropunctatin and monascorubrin)and red pigments(rubropunctamine and monascorubramine)were extremely sensitive to solvent pH.With the increase of pH,the spectra of orange pigments showed a complex red shift;the more red shift was acquired when solvent pH was higher;the color characteristics of the orange pigment solution shifted continuously from yellow to purple,passing from orange and yellow.With the increase of pH,the spectra of red pigments showed a blue shift;the color characteristics of the red pigment solution shifted from red to light red.However,the spectra and color characteristics of yellow pigments(monascin and ankaflavin)and water-soluble red pigments(N-glutarylrubropunctamine and N-glutarylmonascorubramine)were not sensitive to solvent pH.Their visible spectra and color characteristics were very stable under all investigated pH.These results can provide reference for the application of Monascus pigments based on solvent pH.The amination reaction of orange pigments with amino compounds was also sensitive to solvent pH.With the increase of pH,the amination reaction rate increased significantly and the orange pigments were gradually converted to the corresponding red pigments.In addition,the pH-sensitive spectral properties could seriously impact the content analysi of Monascus pigments based on the absorbance at specific wave length and the composition analysis of Monascus pigments based on the spectral properties.Based on the presented results,we propose that the sample preparation and analysis of Monascus pigments should be uniformly performed at low pH(? 2.5)to avoid variations of spectra and the creation of artefacts due to the occurrence of amination reactions,and ensure an accurate analysis that truly reflects pigment characteristics in the samples and differences among the samples.(2)The photostability of Monascus pigments were closely related to solvent pH.Orange pigments(rubropunctatin and monascorubrin)and red pigments(rubropunctamine and monascorubramine)have better photostability at p H 2.5.But with the increase of solvent pH,their photodegradation rate increased significantly and their color characteristics changed obviously during photodegradation.Compared with orange and red pigments,yellow pigments(monascin and ankaflavin)and water-soluble red pigments(N-glutarylrubropunctamine and Nglutarylmonascorubramine)showed better photostability at all investigated pH,their photodegradation rate was relatively slow,and their color characteristics changed little during photodegradation.Therefore,yellow pigments and the water-soluble red pigments have wide application considering solvent pH and good photostability and stable color charateristics at all pH investigated.Orange pigments and red pigments were suitable to be used at low pH to keep good photostability and stable color charateristics.(3)Monascus pigment composition during fermentation was synergistically regulated by pH and nitrogen sources.A large amount of intracellular pigments dominated by orange pigments were produced at low pH,independently of the nitrogen source employed.At high pH,when ammonium sulfate was used as nitrogen source,the pigments were mainly intracellular and the intracellular pigments were dominated by red pigments.Conversely,when peptone was used,intracellular pigments were dominated by yellow pigments and a large amount of extracellular water-soluble red pigments were produced.Color characteristics of the pigment samples depended on the major pigment component.Therefore,in fermentation of Monascus pigments,the targeted pigment component could be accumulated heavily and pigments with desirable color characteristics could be achieved through p H and nitrogen source control.(4)Under low pH,submerged fermentation with Monascus anka GIM 3.592 mainly accumulated intracellular orange pigments(rubropunctatin and monascorubrin)and yellow pigments(monascin and ankaflavin),however,extractive fermentation in nonionic surfactant micelle aqueous solution was found to not only export intracellular orange and yellow pigments to the extracellular broth,but also to form four other yellow pigments that accounted for a large proportion of the extracellular pigments and were not produced in submerged fermentation.The four new yellow pigments presented identical UV-Vis spectra with absorption maxima at 430 nm and identical fluorescence spectra with absorption maxima(emission)at 565 nm.They had strong orange fluorescence under specific excitation wavelengths.Direct extract with Triton X-100 aqueous solution could not produce the new yellow pigments and could only extract the intracellular orange and yellow pigments into the extracellular broth.Through comparing the difference in molecular weight between the new yellow pigments and orange pigments and the accumulation of each pigment during different extractive fermentation process,it indicated that the new yellow pigments might be formed by bioconversion of orange pigments during the exportation process.The yield of extracellular pigments increased with the increase in Triton X-100 concentration.Also,the yield of extracellular pigments was impacted by the feeding time of Triton X-100,and feeding Triton X-100 at the middle stage of fermentation gave the highest yield of extracellular pigments.(5)Extractive fermentation and immobilized cell fermentation could be combined successfully to continuously produce Monascus yellow pigments extracellularly.Extractive fermentation with immobilized cell could give the shorter fermentation period and increase the cell density.The immobilized cell could be reused at least seven batches.In repeated-batch extractive fermentation with immobilized cells,the mean productivity reached up to 22.31 AU410 nm/day within the first four batches(13 days)and the cumulative yield was 289.97 AU410 nm;within seven batches(25 days),the mean productivity reached up to 19.70 AU410 nm/day and the cumulative yield was 492.53 AU410 nm.