Enhancement of microbial pigment production from Monascus ruber by sodium octanoate addition.

BACKGROUND: The addition of fatty acids and other molecules to culture media may intensify the production of biomolecules, such as monascus pigments, however, few studies of this have been developed. Thus, the objective of the present study was to investigate the effects of adding sodium octanoate to the culture medium, with a view to increasing the synthesis and production of the pigments produced by Monascus ruber CCT 3802 on solid and submerged cultivations. METHODS: Monacus ruber CCT 3802 was cultivated on solid and submerged media supplemented with different concentrations of sodium octanoate. The radial growth rate of the colonies was obtained from the declivity of the linear regression of the radius of the colonies as a function of cultivation time and the kinetics of submerged cultivations were performed. The filtrate obtained was submitted to scanning spectrophotometry at a range from 350 to 550 nm and the color parameters were determined by using the CIELAB color system. The data were submitted to a univariate analysis of variance (ANOVA) and the means obtained for each treatment submitted to Tukey's test using Statistica version 5.0 software at a 5% level of significance. RESULTS: Sodium octanoate exerted a strong influence on growth and pigment production in solid and submerged cultivations. The values for L*, a* and b* were positive for pigments produced, with regards to colors close to red and yellow. In the media supplemented with 1.0 mM and 1.5 mM of sodium octanoate, the production of red pigments became expressive from 48 hours-cultivation, increasing considerably from the second to the fourth days. This shows that supplementation with sodium octanoate provides a greater production of pigments in a shorter time interval than the control culture, which required 144 hours of cultivation to present a higher value for AU510nm, which directly influenced pigment productivity. CONCLUSIONS: The addition of sodium octanoate exerted a significant influence on both microbial growth and pigment production in both solid and submerged cultivations. The supplementation of the submerged cultures with sodium octanoate was responsible for an expressive production of pigments in just 48 hours, whereas 144 hours were necessary in the absence of sodium octanoate. These results are promising for increasing the productivity of pigment production, including possibilities for application on an industrial scale.

Quantitative Proteomics Analysis by Sequential Window Acquisition of All Theoretical Mass Spectra-Mass Spectrometry Reveals a Cross-Protection Mechanism for Monascus To Tolerate High-Concentration Ammonium Chloride.

To achieve the accumulation of targeted secondary metabolites, microorganisms must adopt various protection mechanisms to avoid or reduce damage to cells caused by abiotic stresses, which formed from the changes of physical and chemical culture conditions. The protection mechanism of Monascus sp. to tolerate high-concentration ammonium chloride was analyzed by sequential window acquisition of all theoretical mass spectra-mass spectrometry proteomics in this work, and the results indicated that abiotic stresses caused by high-concentration ammonium chloride inhibited the synthesis of chitin and glycoprotein, leading to a decrease in cell wall integrity and, thus, affecting cell growth. At the same time, it also inhibited the complex enzyme III and IV activities of the mitochondrial cytochrome respiratory chain, leading to an increase in reactive oxygen species (ROS) levels. With the aim to respond to abiotic stresses, the cross-protection mechanism was implemented in Monascus, including self-protection of the Monascus cell by promoting synthesis of trehalose, a molecular chaperone that facilitates protein folding (such as heat-shock protein) and autophagy-related proteins, through not the enzyme protection system (superoxide dismutase, peroxidase, catalase, NADPH oxidase, and alternative oxidase) but the glutathione/glutaredoxin system, to maintain the intracellular redox state and then eliminate or reduce ROS damage to the cell. At the same time, an alternative respiratory pathway related to NADH dehydrogenase was activated to balance the material and energy metabolism.

Effect of nonionic surfactant Brij 35 on morphology, cloud point, and pigment stability in Monascus extractive fermentation.

BACKGROUND: Nonionic surfactant Brij 35 in submerged fermentation of Monascus can significantly increase Monascus pigment yield. Here, the effects of nonionic surfactant Brij 35 on Monascus pigment secretion in extractive fermentation are discussed in terms of cell morphology, cloud point change, and pigment stability. RESULTS: At Brij 35 concentrations up to 32 g L-1 , the higher concentrations led to the loosening of the network structure on the surface of the fungal wall, enhanced cell wall permeability, and increased abundance of lipid droplets. Alternatively, when the concentration of Brij 35 exceeded 32 g L-1 , a large amount of substances accumulated on the surface of the fungal wall, permeability reduced, and the degree of oil droplet dispersion in cells decreased. Further, during extractive fermentation, Brij 35 induced formation of a grid structure on the fungal wall surface beginning on day 2, increased the number of intracellular lipid droplets, and promoted intracellular pigment secretion into the extracellular environment. When the cloud point temperature in the fermentation system approached that of fermentation, the nonionic surfactant exhibited stronger Monascus pigment extraction capacity, thereby enhancing pigment yield. Hence, Brij 35 can improve pigment stability and effectively reduce damage caused by natural factors, such as light and temperature. CONCLUSION: Brij 35 promotes the secretion of pigment by changing the fungal wall structure and cloud point, as well as by improving pigment stability. © 2020 Society of Chemical Industry.

Evaluation of tyrosol and farnesol as inducer in pigment production by Monascus purpureus ATCC16365.

This study focused on investigating the effect of exogenously applied two quorum sensing molecules (tyrosol and farnesol) on the synthesis of bioactive metabolites (pigments, lactic acid, ethanol, and citric acid) in Monascus purpureus ATCC16365. None of the tested concentrations (62.5, 125, 250, and 500 µl/L) of farnesol affected the synthesis of metabolites as well as cell growth. As with farnesol application, none of the tested concentrations (3.45, 6.9, 13.8, and 27.6 mg/L) of tyrosol caused a significant change in the synthesis of lactic acid and citric acid as well as cell growth. Conversely, all of the tested concentrations of tyrosol increased pigment synthesis but reduced ethanol synthesis, compared with the control. Maximum increases (3.16-, 2.68-, and 2.87-fold increase, respectively) in yellow, orange, and red pigment production were achieved, especially when 6.9-mg/L tyrosol was added to the culture on day 3. On the contrary, 6.9-mg/L tyrosol reduced the content of citrinin by approximately 51.5%. This is the first report on the effect of tyrosol and farnesol on the synthesis of Monascus metabolites. Due to potential properties, such as low price, nonhuman toxicity, promotion of pigment synthesis, and reduction in citrinin synthesis, tyrosol can be used as a novel inducer in the fermentative production of Monascus pigments.

Monasone Naphthoquinone Biosynthesis and Resistance in Monascus Fungi.

Despite the important biological activities of natural product naphthoquinones, the biosynthetic pathways of and resistance mechanisms against such compounds remain poorly understood in fungi. Here, we report that the genes responsible for the biosynthesis of Monascus naphthoquinones (monasones) reside within the gene cluster for Monascus azaphilone pigments (MonAzPs). We elucidate the biosynthetic pathway of monasones by a combination of comparative genome analysis, gene knockouts, heterologous coexpression, and in vivo and in vitro enzymatic reactions to show that this pathway branches from the first polyketide intermediate of MonAzPs. Furthermore, we propose that the monasone subset of biosynthetic genes also encodes a two-tiered resistance strategy in which an inducible monasone-specific exporter expels monasones from the mycelia, while residual intracellular monasones may be rendered nontoxic through a multistep reduction cascade.IMPORTANCE The genes for Monascus naphthoquinone (monasone) biosynthesis are embedded in and form a composite supercluster with the Monascus azaphilone pigment biosynthetic gene cluster. Early biosynthetic intermediates are shared by the two pathways. Some enzymes encoded by the supercluster play double duty in contributing to both pathways, while others are specific for one or the other pathway. The monasone subcluster is independently regulated and inducible by elicitation with competing microorganisms. This study illustrates genomic and biosynthetic parsimony in fungi and proposes a potential path for the evolution of the mosaic-like azaphilone-naphthoquinone supercluster. The monasone subcluster also encodes a two-tiered self-resistance mechanism that models resistance determinants that may transfer to target microorganisms or emerge in cancer cells in case of naphthoquinone-type cytotoxic agents.

Effects of glutamic acid on the production of monacolin K in four high-yield monacolin K strains in Monascus.

Monascus purpureus is a traditional Chinese microbe that can be used as a medicinal herb and is edible. To improve the yield of monacolin K, we optimized the medium of M. purpureus with high-yield monacolin K strains. When high-yield strains C8, D8, E3, and I1 were grown in glutamic medium instead of the original medium, monacolin K production was increased. Among these strains, C8 exhibited the highest monacolin K production in glutamic acid medium, with levels increased 4.80-fold. RT-qPCR demonstrated that glutamic acid enhanced the expression of mokC and mokG. Observation of Monascus mycelium morphology using SEM showed that mycelia exhibited more folds, swelling, curves, and fractures. Thus, glutamic acid may promote the growth of the mycelium and appeared to increase the permeability of the cell membrane. This lays a foundation for research on the regulatory effect of glutamic acid and provides a theoretical basis for the industrialization and commercialization of Monascus.

NaCl Inhibits Citrinin and Stimulates Monascus Pigments and Monacolin K Production.

Applications of beneficial secondary metabolites produced by Monascus purpureus (M. purpureus) could be greatly limited for citrinin, a kidney toxin. The link of NaCl with cell growth and secondary metabolites in M. purpureus was analyzed with supplementations of different concentrations of NaCl in medium. The content of citrinin was reduced by 48.0% but the yellow, orange, red pigments and monacolin K productions were enhanced by 1.7, 1.4, 1.4 and 1.4 times, respectively, compared with those in the control using NaCl at 0.02 M at the 10th day of cultivation. NaCl didn't affect the cell growth of M. purpureus. This was verified through the transcriptional up-regulation of citrinin synthesis genes (pksCT and ctnA) and the down-regulation of the Monascus pigments (MPs) synthesis genes (pksPT and pigR). Moreover, the reactive oxygen species (ROS) levels were promoted by NaCl at the 2nd day of cultivation, and then inhibited remarkably with the extension of fermentation time. Meanwhile, the activities of superoxide dismutase (SOD) and catalase (CAT), and the contents of total glutathione (T-GSH) were significantly enhanced in the middle and late stages of cultivation. The inhibition effect on colony size and the growth of aerial mycelia was more obvious with an increased NaCl concentration. Acid and alkaline phosphatase (ACP and AKP) activities dramatically increased in NaCl treatments. NaCl could participate in secondary metabolites synthesis and cell growth in M. purpureus.

Effects of rutin and its derivatives on citrinin production by Monascus aurantiacus Li AS3.4384 in liquid fermentation using different types of media.

The mycotoxin citrinin is often produced during fermentation of Monascus products. We studied the effects of flavonoids on citrinin production by Monascus aurantiacus Li AS3.4384 (MALA) by adding rutin, α-glucosylrutin, or troxerutin to the fermentation medium, in a first-of-its-kind study. Appropriate amounts of rutin, α-glucosylrutin, or troxerutin did not affect normal mycelial growth. Addition of 5.0 g/l of rutin only weakly reduced (29.2%) citrinin production, relative to inhibition by 5 g/l α-glucosylrutin or troxerutin (by 54.7% and 40.6%, respectively). In starch inorganic liquid culture media, addition of 20.0 g/l of troxerutin, followed by fermentation for 12 days, reduced citrinin yield by 75.26%. Addition of 15.0 g/l of troxerutin to low-starch peptone liquid fermentation media reduced citrinin yield by 87.9% after 14 days of fermentation, and addition of 30.0 g/l troxerutin to yeast extract sucrose liquid media for 12 days reduced citrinin yield by 53.7%.

Effects of hurdle technology on Monascus ruber growth in green table olives: a response surface methodology approach

ABSTRACT An ascomycetes fungus was isolated from brine storage of green olives of the Arauco cultivar imported from Argentina and identified as Monascus ruber. The combined effects of different concentrations of sodium chloride (3.5-5.5%), sodium benzoate (0-0.1%), potassium sorbate (0-0.05%) and temperature (30-40 °C) were investigated on the growth of M. ruber in the brine of stored table olives using a response surface methodology. A full 24 factorial design with three central points was first used in order to screen for the important factors (significant and marginally significant factors) and then a Face-Centered Central Composite Design was applied. Both preservatives prevented fungal spoilage, but potassium sorbate was the most efficient to control the fungi growth. The combined use of these preservatives did not show a synergistic effect. The results showed that the use of these salts may not be sufficient to prevent fungal spoilage and the greatest fungal growth was recorded at 30 °C.

Analyses of Monascus pigment secretion and cellular morphology in non-ionic surfactant micelle aqueous solution.

Monascus pigments produced by Monascus spp. are widely used as natural food colourants. Extractive fermentation technology can facilitate the secretion of intracellular Monascus pigments into extracellular non-ionic surfactant micelle aqueous solution, so as to avoid the feedback inhibition and decomposition. In this study, behaviour of the trans-membrane secretion of Monascus pigments was investigated using morphological and spectroscopic analyses. Laser scanning confocal microscopy (LSCM) traced that pigment secretion occurred through rapid trans-membrane permeation in 4 min, with a simultaneous conversion in pigment characteristics. Approximately 50% of intracellular pigments (AU470 ) extracted to extracellular broth with 40 g l-1 Triton X-100, indicating the capacity for pigment extraction was limited by the saturation concentrations of surfactant. Scanning electron microscope (SEM) and transmission electron microscope (TEM) imaging showed some damage in the cell wall but an intact cell membrane with a slightly increased mycelial diameter. However, the physiological properties of the cell membrane, including integrity, fluorescence intensity and permeability, were altered. A diagram was provided to demonstrate the behaviour of Monascus pigment secretion induced by Triton X-100. This study lays a foundation for the further investigation of Monascus pigment metabolism and secretion in extractive fermentation.

Effects of hurdle technology on Monascus ruber growth in green table olives: a response surface methodology approach.

An ascomycetes fungus was isolated from brine storage of green olives of the Arauco cultivar imported from Argentina and identified as Monascus ruber. The combined effects of different concentrations of sodium chloride (3.5-5.5%), sodium benzoate (0-0.1%), potassium sorbate (0-0.05%) and temperature (30-40°C) were investigated on the growth of M. ruber in the brine of stored table olives using a response surface methodology. A full 24 factorial design with three central points was first used in order to screen for the important factors (significant and marginally significant factors) and then a Face-Centered Central Composite Design was applied. Both preservatives prevented fungal spoilage, but potassium sorbate was the most efficient to control the fungi growth. The combined use of these preservatives did not show a synergistic effect. The results showed that the use of these salts may not be sufficient to prevent fungal spoilage and the greatest fungal growth was recorded at 30°C.

Fluconazole treatment enhances extracellular release of red pigments in the fungus Monascus purpureus.

Traditional methods for the production of food grade pigments from the fungus Monascus spp. mostly rely on submerged fermentation. However, the cell-bound nature and intracellular accumulation of pigments in Monascus spp. is a major hurdle in pigment production by submerged fermentation. The present study focused on the investigation of the effect of the antifungal agent fluconazole on red pigment production from Monascus purpureus (NMCC-PF01). At the optimized concentration of fluconazole (30 µg ml-1), pigment production was found to be enhanced by 88% after 96 h and it remained constant even after further incubation up to 168 h. Ergosterol, a sterol specific to fungi, was also extracted and estimated as a function of fungal growth. The concentration of ergosterol in fluconazole-treated fermentation broth was reduced by 49% as compared to control broth. Thus it could be responsible for facilitating the release of intracellular and cell-bound pigments. Nevertheless, the role of cell transporters in transporting out the red pigments cannot be ignored and deserves further attention. Qualitative analysis of red pigment by thin layer chromatography, UV spectroscopy and mass spectrometric analysis (ESIMS) has confirmed the presence of the well-known pigment rubropunctamine. In addition, this fermentation process produces citrinin-free pigments. This novel approach will be useful to facilitate increased pigment production by the release of intracellular or cell-bound Monascus pigments.

Investigation of relationship between lipid and Monascus pigment accumulation by extractive fermentation.

Fermented Monascus pigments have been utilized as traditional Chinese medicine and food colorant for thousands of years. Under the limited nitrogen concentration and/or low initial pH 2.5 conditions, it was observed that production of intracellular pigments and accumulation of microbial lipids (high content reaching to approximately 50% in dry cell weight) by edible Monascus anka exhibited a positive correlated relationship. Extractive fermentation in nonionic surfactant micelle aqueous solution selectively exported the intracellular Monascus pigments into its extracellular broth, in which the concentration of intracellular pigments was negligible while the extracellular one was enhanced. The extractive fermentation provides a novel strategy for shifting of the metabolic channeling from intracellular lipid accumulation to Monascus pigment production. High pigment concentration, i.e., approximately 40 AU of extracellular Monascus pigments, was achieved by extractive fermentation at a relatively high nonionic surfactant concentration 10 g/l. This phenomenon might be attributed to the nonionic surfactant micelles acting as pigment reservoirs by biomimetic of intracellular lipids.

Effect of oxygen supply on Monascus pigments and citrinin production in submerged fermentation.

The influence of oxygen supply on Monascus pigments and citrinin production by Monascus ruber HS.4000 in submerged fermentation was studied. For Monascus cultivation with high pigments and low citrinin production, the initial growth phase, mid-stage phase, and later-stage production phase were separated by shifting oxygen supply. The optimal condition for the fermentation process in shake-flask fermentation was a three-stage rotating rate controlled strategy (0-48 h at 150 rpm, 48-108 h at 250 rpm, 108-120 h at 200 rpm) with medium volume of 100 mL added to 250 mL Erlenmeyer flasks at 30°C for 120 h cultivation. Compared to constant one-stage cultivation (medium volume of 100 mL, rotating rate of 250 rpm), the pigments were reduced by 40.4%, but citrinin was reduced by 64.2%. The most appropriate condition for the fermentation process in a 10 L fermentor is also a three-stage aeration process (0-48 h at 300 L/h, 48-96 h at 500 L/h, 96-120 h at 200 L/h) with agitation of 300 rpm at 30°C for 120 h cultivation, and 237.3 ± 5.7 U/mL pigments were produced in 120 h with 6.05 ± 0.19 mg/L citrinin in a 10 L fermentor. Compared to aeration-constant (500 L/h) cultivation, pigment production was increased by 29.6% and citrinin concentration was reduced by 79.5%.

Enhanced production of Monacolin K by addition of precursors and surfactants in submerged fermentation of Monascus purpureus 9901.

The main problem in Monacolin K (MK) production by submerged fermentation of Monascus purpureus is low productivity. In this study, on one hand, addition of precursors was used to activate the biosynthesis of MK. When 4.0 g/L of sodium citrate was supplemented at the 48th H of the fermentation, the final MK production reached to 1,658.9 ± 28.5 mg/L after 20 day of fermentation, which was improved by 52.6% compared with that of the control. On the other hand, addition of surfactants could increase the permeability of cell membrane, thus driving more intracellular metabolites secreted into the fermentation broth and alleviating the product inhibition. When 40.0 g/L of Triton X-100 was added at the beginning of the fermentation, the final MK production reached to 2,026.0 ± 30.4 mg/L after 20 day of fermentation, which was improved by 84.9% compared with that of the control. These results are helpful to provide some new insights into the biosynthetic regulation on MK production; the approach can be applied to other fungal fermentation processes for enhancing production of useful metabolites.

Enhanced production of pigments by addition of surfactants in submerged fermentation of Monascus purpureus H1102.

BACKGROUND: The production of pigments by Monascus spp. has attracted increasing attention. Modification of the cell membrane structure by addition of surfactants has proved to be effective for the secretion of intracellular metabolites. Hence in this study the effects and underlying mechanism of surfactants on the production of pigments in submerged fermentation of Monascus purpureus H1102 were systematically investigated. RESULTS: Various surfactants exerted significant but different impacts on the biomass and production of pigments. The maximum production of pigment (304.3 U mL(-1) ) and highest extracellular/intracellular pigment ratio (1.46) were achieved when 15 g L(-1) Triton X-100 was added at 24 h of fermentation, corresponding to significant increases of 88.4 and 240% respectively compared with the control. Meanwhile, the concentration of citrinin (0.94 mg L(-1) ) was 20.6% lower than that of the control. A further study on the fatty acid composition of M. purpureus H1102 showed that the unsaturated/saturated fatty acid ratio and the index of unsaturated fatty acid increased significantly with the addition of Triton X-100. CONCLUSION: The addition of surfactant Triton X-100 could greatly enhance the production of pigment. It was suggested that Triton X-100 facilitated the secretion of intracellular pigment and therefore enhanced pigment production accordingly.

Solubilization capacity of nonionic surfactant micelles exhibiting strong influence on export of intracellular pigments in Monascus fermentation.

In this study, perstractive fermentation of intracellular Monascus pigments in nonionic surfactant micelle aqueous solution had been studied. The permeability of cell membrane modified by nonionic surfactant might have influence on the rate of export of intracellular pigments into its extracellular broth while nearly no effect on the final extracellular pigment concentration. However, the solubilization of pigments in nonionic surfactant micelles strongly affected the final extracellular pigment concentration. The solubilization capacity of micelles depended on the kind of nonionic surfactant, the super-molecule assembly structure of nonionic surfactant in an aqueous solution, and the nonionic surfactant concentration. Elimination of pigment degradation by export of intracellular Monascus pigments and solubilizing them into nonionic surfactant micelles was also confirmed experimentally. Thus, nonionic surfactant micelle aqueous solution is potential for replacement of organic solvent for perstractive fermentation of intracellular product.

Effects of cyclic AMP on development and secondary metabolites of Monascus ruber M-7.

AIM: To study effects of cyclic adenosine monophosphate (cAMP) on development and secondary metabolites of Monascus ruber M-7. METHODS AND RESULTS: Plate culture, liquid-state fermentation (LSF) and solid-state fermentation (SSF) were used to evaluate effects of cAMP on colonial growth, spore formation and polyketide production of Strain M-7. The results revealed that the variation trends of colonial sizes, numbers of sexual spores and red pigment contents of M-7 were in a dose-dependent manner. And generally they increased and decreased with cAMP concentrations in the ranges of low cAMP concentrations and high cAMP concentrations, respectively. But the variation trends of numbers of asexual spores and citrinin production in both LSF and SSF were opposite to those of colonial sizes, sexual sporulation and red pigment. CONCLUSIONS: The regulation of cAMP on development and secondary metabolites in Strain M-7 was in a dose-dependent pattern. And red pigment might convert to citrinin under changing cAMP concentrations. SIGNIFICANCE AND IMPACT OF THE STUDY: The effects of cAMP on Strain M-7 in SSF give a new clue to enhance beneficial polyketides and reduce citrinin produced by M. ruber.

Synthesis, antidepressant and antifungal evaluation of novel 2-chloro-8-methylquinoline amine derivatives.

A new series of N-[(2-chloro-8-methylquinolin-3-yl)methyl]-(substituted)-aniline/butylamine/cyclohexylamine/benzylamine derivatives (4a-p) was synthesized by nucleophilic substitution reaction of 2-chloro-3-(chloromethyl)-8-methylquinoline 3 with various aliphatic and aromatic amines in absolute ethanol in the presence of triethylamine (TEA). The newly synthesized secondary amines were characterized by the combined use of IR, 1H NMR, 13C NMR, mass spectral data and microanalyses. The antidepressant activity of the synthesized compounds (4a-p) was evaluated by Forced swim test in rats and their neurotoxicity was evaluated by the rotarod test. Test compounds and clomipramine were administered intraperitoneally at dose of 100 mg/kg and 20 mg/kg respectively. Preliminary antidepressant screening of compounds (4a-p) revealed that compounds 4b, 4c, 4d, 4e, 4i and 4o significantly (P<0.01) reduces the duration of immobility time. These compounds were also tested in-vitro for MAO inhibitory effect. All the compounds were also screened for antifungal activity against Aspergillus niger MTCC 281, Aspergillus flavus MTCC 277, Monascus purpureus MTCC 369 and Penicillium citrinum NCIM 768 strains.

Breeding an amylolytic yeast strain for alcoholic beverage production.

A starch-utilizing, yeast-like fusant was successfully created from fused protoplasts of Schizosaccharomyces pombe and Monascus anka, and the feasibility of using this fusant as a new strain for alcoholic beverage development was reported. The new fusant utilized various carbon sources more efficiently than its parent cells did. Rice koji prepared separately by cultivating the fusant and its parental strains on rice was compared to explore the effect of yeast strain on the production of α-amylase, glucoamylase, and acid protease that are crucial in wine making using cereal grains. It was found that the fusant produced greater levels of the above-mentioned enzymes than its parental strain does. Consequently, the usage of this fusant in the alcoholic fermentation of polished rice was found to reduce approximately 50% consumption of added glucoamylase than when its parental strain was used. Besides, at the end of fermentation, the fusant yeast resulted in a mash with distribution of flavor components very different from that produced by its parental strains. Thus, the fusant can be used as a new yeast strain for creating novel alcoholic beverages.