Extracellular pigment production from Talaromyces purpureogenus under stress conditions and potential of produced pigments as anti-glycation agents

Influence of light wavelengths, light intensity, temperature, and pH on biosynthesis of extracellular and intracellular pigment and biomass of Pseudomonasaeruginosa NR1

Background and objective: Monascus purpureus is a filamentous fungus with ability to produce pigments with therapeutic properties. Red pigments are especially used as additives, antioxidants, preservatives and substitutions for nitrites in food technology. To decrease fermentation costs, agro-industrial wastes such as soybean meals have been used as substrates. In the current study, red pigment production by Monascus purpureus on soybean meals was optimized. Material and methods: In this study, red Monascus pigment production by Monascus purpureus ATCC 16362 was carried out under submerged fermentation using soybean meals as nitrogen sources to replace yeast extracts. Central composite design was used to assess the optimum level of soybean meal replacement (0-100%), ZnSO4.7H2O concentration (0-0.02 g l-1) and thermal stress time of spore suspension at 70°C (50-90 s). Red Monascus pigment and biomass productions were assessed as dependent responses. Results and conclusion: The maximum production of red Monascus pigment (4.54 AU ml-1) was achieved under conditions of soybean meal replacement of 79.72%, ZnSO4.7H2O concentration of 0-0.02 g l-1 and thermal stress time of spore suspension of 81.89 s. The average yield of red Monascus pigment, conversion factor of biomass in red pigment YP/X and cell productivity included 0.324 AU ml-1 day-1, 1.10 AU L g-1 and 0.292 g l-1 day-1, respectively. Results of the current study have demonstrated that combination of soybean meal and yeast extract as nitrogen source is beneficial for the production of red Monascus pigment by Monascus purpureus. Conflict of interest: The authors declare no conflict of interest.

Maltose syrup residue as the substrate for Monascus pigments production

Pigment production from filamentous fungi is gaining interest due to the diversity of fungal species, the variety of compounds synthesized, and the possibility of controlled massive productions. The Talaromyces species produce a large panel of metabolites, including Monascus-like azaphilone pigments, with potential use as natural colorants in industrial applications. Optimizing pigment production from fungal strains grown on different carbon and nitrogen sources, using statistical methods, is widespread nowadays. The present work is the first in an attempt to optimize pigments production in a culture of the marine-derived T. albobiverticillius 30548, under the influence of several nutrients sources. Nutrient combinations were screened through the one-variable-at-a-time (OVAT) analysis. Sucrose combined with yeast extract provided a maximum yield of orange pigments (OPY) and red pigments (RPY) (respectively, 1.39 g/L quinizarin equivalent and 2.44 g/L Red Yeast pigment equivalent), as well as higher dry biomass (DBW) (6.60 g/L). Significant medium components (yeast extract, K2HPO4 and MgSO4·7H2O) were also identified from one-variable-at-a-time (OVAT) analysis for pigment and biomass production. A five-level central composite design (CCD) and a response surface methodology (RSM) were applied to evaluate the optimal concentrations and interactive effects between selected nutrients. The experimental results were well fitted with the chosen statistical model. The predicted maximum response for OPY (1.43 g/L), RPY (2.59 g/L), and DBW (15.98 g/L) were obtained at 3 g/L yeast extract, 1 g/L K2HPO4, and 0.2 g/L MgSO4·7H2O. Such optimization is of great significance for the selection of key nutrients and their concentrations in order to increase the pigment production at a pilot or industrial scale.

A hyperpigment-producing mutant, R-10847, was derived from Monascus kaoliang F-2 (ATCC 26264) through a series of mutagenesis steps. The mutant produced a large quantity of Monascus pigment when grown in mantou (steamed bread) by solid culture. The mutant produced pigments extracellularly by extruding the pigments outside the cell in a lump together with some viscous substances. The productivity of pigment was about 100-fold greater than that of the wild type. The mutant lost the capability of spore formation, the growth rate decreased, and both the size and quantity of conidia were reduced. The color of the pigment produced by the mutant changed from orange to deep red.

Kinetic analysis of red pigment and citrinin production by Monascus ruber as a function of organic acid accumulation.

The competence of the living creatures to sense and respond to light is well known. The effect of darkness and different color light quality on biomass, extracellular and intracellular pigment yield of five potent pigment producers Monascus purpureus, Isaria farinosa, Emericella nidulans, Fusarium verticillioides and Penicillium purpurogenum, with different color shades such as red, pink, reddish brown and yellow, were investigated. Incubation in total darkness increased the biomass, extracellular and intracellular pigment production in all the fungi. Extracellular red pigment produced by M. purpureus resulted maximum in darkness 36.75 + or - 2.1 OD and minimum in white unscreened light 5.90 + or - 1.1 OD. Similarly, intracellular red pigment produced by M. purpureus resulted maximum in darkness 18.27 + or - 0.9 OD/g and minimum in yellow light 8.03 + or - 0.6 OD/g of substrate. The maximum biomass production was also noticed in darkness 2.51 g/L and minimum in yellow light 0.5 g/L of dry weight. In contrast, growth of fungi in green and yellow wavelengths resulted in low biomass and pigment yield. It was found that darkness, (red 780-622 nm, blue 492-455 nm) and white light influenced pigment and biomass yield.

Marine-derived fungi that inhabit severe changing environments have gained increasing interest for their ability to produce structurally unique natural products. Fungi belonging to the Talaromyces and the close Penicillium genera are among the most promising microbes for bioactive compound production, including colored metabolites. Coupling pigment producing capability with bioactive effectiveness would be a valuable challenge in some specific fields such as dyeing, cosmeceutical, or food industries. In this sense, Talaromyces albobiverticillius 30548, a red pigment producing strain, has been isolated from the marine environment of Reunion Island, Indian Ocean. In this research, we analyzed the effect of temperatures (21–27 °C) and salinity levels (0–9%) on fungal growth and pigment production. Maximum pigment yield was obtained in non-salted media, when cultured at 27 °C after 10 days of submerged fermentation in PDB. However, maximum dry biomass production was achieved at stressed condition with 9% sea salts concentrated media at the same temperature. The results indicate that salinity of the culture media positively influences the growth of the biomass. Inversely, pigment production decreases with increase in salinity over 6%. Color coordinates of secreted pigments were expressed in CIELAB color system. The hue angles (h°) ranged from red to yellow colors. This indicated that the color distribution of fungal pigments depends on the salinity in the culture media. This study emphasizes the impact of abiotic stress (salt and temperature) on the growth and metabolome of marine-derived fungal strains.

The effect of different levels of salt, sodium nitrite, polyphosphate and various sugars on growth, pigment production, protease activity and culture pH caused by Monascus purpureus was studied in broth medium and ground meat. The addition of sodium chloride (> 50.0 g l(-1)) and polyphosphate (> 3.0g l(-1)) to broth medium decreased mycelial growth, pigment production and protease activity of M. purpureus, whereas low concentrations of sodium nitrite (< 0.2 g l(-1)) promoted mycelial growth and pigment production. When the basal medium and ground meat contained salt, 150.0 g l(-1), the mould growth was stopped. The medium with fructose as carbon source proved to be the most suitable for mycelium growth and pigment production, with maltose and glucose being the second most productive. When sucrose and lactose were used as carbon sources, mycelium growth and pigment production were inhibited but the protease activity increased significantly. The mould showed more tolerance to salt and polyphosphate in ground meat than in broth medium and used sucrose as a carbon source as well as glucose for growth and pigment production in the meat mixture.

Valorization of pineapple peel waste for fungal pigment production using Talaromyces albobiverticillius: Insights into antibacterial, antioxidant and textile dyeing properties

The effect of pH and nitrogen source on pigment production by Monascus purpureus 192F using glucose as the carbon and energy source, was studied in pH-controlled, batch fermentor cultures using HPLC analysis to determine individual pigment concentrations. A maximum of four pigments were detected in fungal extracts. These were the yellow pigments monascin and ankaflavin, the orange rubropunctatin and the red pigment monascorubramine. Monascorubramine was present as the major product in all instances. Fungal growth and ankaflavin synthesis were favoured at low pH (pH 4.0), whereas production of the other pigments was relatively independent of pH. The nature of the nitrogen source affected fungal growth and pigment production, independent of pH. Ammonium and peptone as nitrogen sources gave superior growth and pigment concentrations compared to nitrate. Ankaflavin was not detected in nitrate cultures. The highest red pigment production was obtained using a glucose-peptone medium at pH 6.5, due to the secretion of red pigments into the medium under these conditions.

Potato pomace, generated from starch‐processing industry, was applied as a cost‐effective resource for producing Monascus pigments via submerged fermentation. First, the pigment‐production capacity of potato pomace and its hydrolysate was compared. The results indicated that potato pomace was superior to its hydrolysate when used for producing Monascus pigments. The red and yellow pigments produced in potato pomace medium reached 27.8 and 19.7 OD units/ml in 7 days, with the yield of total pigments at 1,187.5 OD units/g, respectively, increased by 127.9%, 19.4%, and 46.3% compared with the data obtained from hydrolysate. Meanwhile, the citrinin produced in potato pomace medium decreased by 22.6%. Afterward, potato pomace, without hydrolysis, was used as carbon source to obtain the optimal pigment production conditions. In the batch fermentation process, it was found that high amount of pomace inhibited the growth rate of mycelia and the productivity of pigments, and the fed‐batch fermentation process could enhance the yield and productivity of pigments. With the same final amount of pomace (80 g/L), the maximal levels of total pigments and productivity obtained from fed‐batch process reached 118.8 OD units/ml and 13.2 OD units/(ml·day), which presented an increase of 35.2% and 67.1% compared with the not fed‐batch group, respectively. The results demonstrated that potato pomace was a cost‐effective substrate for producing Monascus pigments in terms of pigment production capacity and productivity when fed‐batch submerged fermentation was applied.

A red-pigmented bacterial strain was isolated from indoor cultures of the marine microalga Tetraselmis suecica Butcher (Prasynophyceae) and identified as belonging to the genus Alteromonas (strain CECT 4800). Pigment composition was analyzed by means of high-performance liquid chromatography (HPLC), and spectral characteristics of three major pigments with reddish and yellowish colorations were determined. Changes in pigment composition during growth in liquid medium containing either ferric citrate, sodium citrate, glucose or glucose plus iron, as well as in relation to the oxygen concentration in the liquid medium were investigated. Pigment production was found to be enhanced with culture aging, the highest enhancement occurring in iron-deprived cultures. An increase in the major pigment (a red pigment) content of 70% in relation to the initial one took place when a culture fed with ferric citrate was bubbled with molecular oxygen. In contrast, red pigment content decreased to about 50% of the initial content when the culture was sparged with nitrogen. Other pigments did not exhibit substantial modifications. Increase in the red pigment content was also observed after the different cultures were exposed to diverse oxygen concentrations for one hour. Similar results were obtained in cultures submitted to both strong aeration and agitation (5 mL min–1 air and 500 rpm, respectively). Findings of this study suggest that pigment production by Alteromonas sp. (strain CECT 4800), in particular that of the red pigment, could rely on the oxygen concentration in the culture medium due to antioxidative reactions.

Demand for microbial colorants is now becoming a competitive research topic for food, cosmetics and pharmaceutics industries. In most applications, the pigments of interest such as polyketide-based red pigments from fungal submerged cultures are extracted by conventional liquid–liquid extraction methods requiring large volumes of various organic solvents and time. To address this question from a different angle, we proposed, here, to investigate the use of three different aqueous two-phase extraction systems using either ammonium- or imidazolium-based ionic liquids. We applied these to four fermentation broths of Talaromyces albobiverticillius (deep red pigment producer), Emericella purpurea (red pigment producer), Paecilomyces marquandii (yellow pigment producer) and Trichoderma harzianum (yellow-brown pigment producer) to investigate their selective extraction abilities towards the detection of polyketide-based pigments. Our findings led us to conclude that (i) these alternative extraction systems using ionic liquids as greener extractant means worked well for this extraction of colored molecules from the fermentation broths of the filamentous fungi investigated here; (ii) tetrabutylammonium bromide, [N4444]Br-, showed the best pigment extraction ability, with a higher putative affinity for azaphilone red pigments; (iii) the back extraction and recovery of the fungal pigments from ionic liquid phases remained the limiting point of the method under our selected conditions for potential industrial applications. Nevertheless, these alternative extraction procedures appeared to be promising ways for the detection of polyketide-based colorants in the submerged cultures of filamentous fungi.

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. 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. 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. 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.