Pigments Metabolism And Behavior Of Trans-membrane Transport In High Density Extractive Fermentation Of Monascus Spp.

Monascus pigments,as the secondary metabolites with polyketide structures synthesized by Monascus fungi,are consisting of three colors of red,orange,and yellow components.It has been widely used as nature,nutrient,safe,and colorful pigment additives in food and pharmaceutical industries.This paper is aimed at the key problems of the high productivity of Monascus pigment in tranditional submerged fermentation to explore new efficient fermentation thechologies such as high-cell density fermentation,extractive fermentation,and continuous fermentation,and study the pigments metabolism and behavior of trans-membrane transport in high-density extractive fermentation of Monascus anka GIM 3.592.The characteristics and productivity of pigments shifting in high cell density culture of Monascus spp.were mainly investigated.The mechanism of extractive fermentation and the distribution of intracellular and extracellular pigment during were exploded.High-density extractive fermentation and continuous extractive fermentation were studied to improve the pigment productivity.Meanwhile,the correlation between mycelium morphology and pigment production was analyzed in extractive fermentation.Based on these facts,the localization of pigment accumulation,the trans-membrane transport behavior of intracellular pigment and its transformation mechanism in extractive fermentation were investigated accordingly.Additionally,the regulation mechanism of pigment metabolism,the separation characteristics of pigment and the extraction ability of recovered Triton X-100 from extractive fermentation were also studied.The major conclusions are as follow:?1?Achieving the metabolic regulation of high cell density,high proportion of yellow pigment and non-citrinin.The biomass reached 30 g/L by one-step fermentation with high glucose concentration,but high-glucose environment limited the pigment metabolism.High yield of Monascus mycelia up to 39.77g/L was achieved in fed-batch fermentation,but the pigment production was non-coupled with growth at the late stage.Feeding carbon and nitrogen sources were favorable for cell growth but no effect on pigment mebolism in high-density fermentation,while metal elements could both facilitate cell growth and control pigment synthesis.Yellow pigments accumulated constantly at the late stage of fed-batch culture,which resulted in the characteristic absorption peak of intracellular pigments shifted from 430 nm to 410 nm.Non-citrinin generated to ensure the safety of product.?2?Constructing the extractive fermentation system to avoid feedback inhibition,and finding the extraction mechanism.The nonionic surfactant Triton X-100 showed excellent biocompatibility and extraction performance in extractive fermentation with a pigment high-yield strain.The addition of appropriate concentration?40 g/L?of surfactant in the logarithmic stage could maintain cell growth properly and promote pigment metabolism.Triton X-100 owned a characteristic of extraction saturation and the spectrum characteristic of pigments was varied.New yellow pigment components detected by HPLC were generated in micelle aqueous solution and SDS-PAGE revealed the intracellular proteins were also extracted to outside.It showed that the orange pigments were converted to yellow ones with the enzyme catalysis during trans-membrane transport process.?3?Regulating high-density extractive and continuous extractive fermentation to improve pigment metabolism.The biomass could be achieved 20 g/L in high-density extractive fermentation.Total pigment yield improved 17%and extracellular pigment production increased by 162 AU,compared with tranditional batch fermentation.Further optimization showed an appropriate extractive time point(on the 5^th day)and feeding nutrients?fermentation medium?were much more conducive to cell growth and pigment metabolism in fed-batch extractive fermentation.Moreover,the biomass reached 35 g/L and the extracellular pigment was stable at about 100 AU₄₁₀ through semi-continuous extractive fermentation.Through a 30-day continuous extractive fermentation,the average daily productivity for total pigments reached 74.9 AU/day with an increase by 32.6%and 296.3%compared to that in a6-day conventional batch fermentation and a 16-day fed-batch extractive fermentation,respectively.At the meantime,proportions?in total pigments yield?of extracellular pigments was increased gradually from 2.7%to 71.3%.?4?Based on the mechanism and characteristics of extractive fermentation,eatablishing the evaluation method of pigment metabolism via mycelial morphologh.The physiological status in terms of hyphal and pellet diameters exhibited an excellent correlation with pigment accumulation,especially the yield of extracellular pigment in extractive fermentation?r>0.85,p<0.05?.Different extraction reagents had distinct effects on the destruction of hyphae and mycelial pellets,which led to the difference of pigment metabolism.Span-20 was the strongest,followed by Triton X-100 and Tween-80.No significant effect on hyphal and pellet diameters in two-stage extractive fermentation,and the cell growth and pigment metabolism were unchanged.During continuous extractive fermentation,the myclia presented periodic changes suggesting a self-regulated to adapte the extractive environment,and the total pigment yield maintained at a high level with a productivity of 72.3 AU/day.?5?From the relationship of pigment metabolism and mycelial morphologh,localizating the repositories of intracellular pigment and tracing the pigment transport process.LSCM imaged the pigment fluorescence was random distribution,and the fluorescence intensity was constantly increased along with the fermentation time,which was highly correlated with the intracellular pigment yields?r>0.90,p<0.05?.TEM reveled that the cytoplasmic vacuole size increased with the fermentation time and also irregular scattered.Moreover,the increasing size of vacuoles was consistent with the fluorescence intensity?r>0.85,p<0.05?.Therefore,vacuoles were acted as repositories for intracellular pigments.Extractive fermentation with Triton X-100 facilitated the metabolic channel shift from lipid to pigment synthesis,resulted in high pigment yields.LSCM imaged the trans-membrane transport of pigment was a rapid equilibrium process in extractive fermentation,and the release of intracellular fluorescence pigment was related to Triton X-100 concentration but independent of the extraction time.?6?Illuminating the trans-membrane transport pathway of pigment and its transformation mechanism.TEM imaged the cell wall was damaged by Triton X-100,which led to a decrease of USFA/SFA and IUFA,and reduced the membrane lipids fluidity.Meanwhile,the protein conformation,the physiological performances including integrity,permeability,and potential of cell membrane were varied,which improved the cell membrane permeability.Combining the detection of intracellular Triton X-100 concentration,it indicated that Triton X-100 were entered in cells to damage the vacuoles structure and embedded the intracellular pigment,then transported through membrane ion channels.Pigment-surfactant micelles?average size of 21nm?were distributed uniformly in the extracellualr broth.A three-fold decrease in the NAD⁺/NADH ratio in mycelia and a more than 200-fold increase in G6PDH activity in extracellular broth occurred,further suggesting that a reduction reaction for pigment conversion from orange pigments to yellow ones occurred in non-aqueous phase solution.?7?Evaluating the cell activity and parsing the mechanism of pigment metabolism in extractive fermentation.Although the cell growth was limited,it had a good bio-catalytic activity for pigment synthesis in the resting culture with cells from extractive fermentation,showing partial growth associated.The transport and conversion of hydrophobic intracellular pigments needed a Triton X-100 environment during the fermentation with extractive culture cells.Compared with the resting cells culture in CC2 system,the rate of relative content of the intracellular orange pigments to yellow pigments?O/Y?and the oxidation-reduction potential?ORP?in extracellular broth was higher in TC2 and EC2 systems.Moreover,the expression levels of the pigment biosynthetic genes MpFasA2?MpFasB2?mppC?mppD?mppB were up-regulated,whereas genes mppE?MpPKS5?mppR1?mppR2 were down-regulated.It indicated that extractive fermentation was modify the pigment biosynthesis pathway and beneficial for intracellular orange pigment metabolism.?8?Achieving the pigment separation and surfactant reutilization in extractive fermentation.Triton X-100 could be well separated through the THMS-Resin method,and the recycled Monascus pigments contained amount of new yellow pigment components owning high antioxidant properties.Using recycled Triton X-100 to repeat extractive fermentation,it showed an excellent ability for cell growth and pigment metabolism,which superior to the[BMIm]PF6 and[BMIm]BF4 methods.Extractive fermentation with the recycled and unused Triton X-100 could significantly promote the pigment biosynthesis,approximately 35%increment of the total pigment yield obtained at the ratio of 4:1.After fifth recovery and repeated extractive fermentation,the recycling efficiency of Triton X-100,the extraction efficiency of extracellular yellow pigment and the metabolism ability of intracellular yellow pigment could be reached 80%,100%,amd 60%,respectively.This study provided a novel tool of high-cell density for extractive microbial fermentation in two-phase aqueous system,which fulfilled the blank of extractive fermentation of small molecules in the cloud point system.It also provided a classic example for microbial fermentation production of Monascus pigments,as well as other non-water-soluble products.