Optimization of Monascus Pigment Production on Date Waste Substrates Using Solid State Fermentation

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.

Several studies has been conducted to economically cultivate the Monascus sp. However, the potential of using stirred drum bioreactor in solid state fermentation (SSF) for Monascus sp. cultivation has been relatively understudied. Oil palm frond (OPF) petiole has been used as a potential substrate due to its nutritional contents and to add more value to local agricultural waste. This study reports the production of red pigment by Monascus purpureus FTC 5357 in a 2.3 L bench top - stirred-drum bioreactor. The fungus was grown on moistened OPF substrate (60 % (w/w)) supplemented with 2% (w/w) of soy meal peptone. The effects of different aeration rates (0.3-1.0 vvm of humidified air), agitation programme (4-8 cycles per day), and substrate load capacity (25-40 % (v/v) of total drum capacity) on red pigment production are reported. Aeration rate showed a positively correlated interaction to red pigment production in which the highest red pigment were produced using1.0 vvm (6.09 AU/g dry solid), and non-aerated culture showed the lowest red pigment production (0.81 AU/g dry solid). The agitation programme was also showing the positive trend of interaction, in which 8 cycles per day showed the highest red pigment production (4.34 AU/g dry solid) and 4 cycles per day agitation showed the lowest red pigment production.The red pigment production was peaked at 30% (v/v) drum loading capacity (5.61 AU/g dry solid) and the lowest at 25% (v/v) (0.89 AU/g dry solid), whereas 40% (v/v) substrate capacity was incapable of being mixed due to low power output of agitating motor. Results suggested that OPF was a potent source of substrate for the cultivating Monascus purpureus using SSF and all 3 factors (aeration, substrate load capacity and agitation programme) were significantly influenced the red pigment production.

The present study underlines a statistically optimized, low cost, effective approach for efficient co-valorization of two non-efficiently utilized, highly accumulated, raw agro-industrial wastes: corn cob and glycerol for co-production of natural biopigments: monascus orange and red pigments by the aid of Monascus purpureus strain ATCC 16436. A three step sequential, statistical modeling approach: one variable at a time (OVAT), Plackett-Burman design (PBD), and central composite design (CCD) was employed to optimize the production of monascus pigments using co-solid state fermentation of the two raw agro-industrial wastes. Corn cob among other carbon sources (e.g., rice grains, sugarcane bagasse, and potato peel) was the most appropriate substrate triggering co-production of orange and red monascus pigments; deduced from OVAT. Glycerol and inoculum size proved to impose significant consequences (P<0.05) on the production of monascus pigments as inferred from PBD. The optimal levels of inoculum size (12 x 1011 spores/mL) and glycerol (2.17 M) did achieve a maximal color value of 133.77 and 108.02 color value units/mL of orange and red pigments, respectively at 30 oC after 10 days; concluded from CCD with an agitation speed of 150 rpm. Present data would underpin the large scale production of monascus pigments using the present approach for efficient exploitation of such biopigments in food, pharmaceutical and textile industries.

Pencillium purpurogenum 8904.12, a red pigment producer, was isolated from soil screened and selected based on the pigment production. The pigment production by P.purpurogenum was optimizedby using factorial design and Response Surface Methodology (RSM) in SSF. Cassava waste is a low cost and nutrient rich substrate used in this study as a substrate. RSM based central composite design was employed to obtain best combination of substrate concentration, inoculum volume, incubation time, initial moisture and initial pH. By the point prediction tool of Design-Expert 8.0, the optimum values of the factors for maximum red pigment production were determined. Under the optimized conditions (substrate concentration 10 g, inoculum volume 5 ml, 15 days incubation time, 50 % initial moisture and initial pH of 6), the red pigment yield was 28.33 colour value units / g of dry fermented substrate which agreed closely with the predicted yield. The model showed that the value of R2 (0.9936) was high and pvalue of interaction of variance was &lt;0.0001. Hence the model can be said to be of highly significant. A significant Increase in red pigment production was achieved using RSM. Thus, utilization of cassava waste for red pigment production in this study could provide the most effective use of cassava resource, and lead to technology of development for its further utilization and value addition.

Objective: The objective of this work was the production of red and yellow pigments by the fungus Monascus purpureus NRRL 1596 using agroindustrial wastes of the cassava production chain. Methods: Solid state fermentation experiments were carried out by using cassava peels and cassava wastewater as substrate for pigment production by Monascus purpureus NRRL 1596. Initial moisture content and the addition of soy bran to improve Carbon/nitrogen ratio were optimized in order to follow the growth kinetics and pigment production in the selected substrate. Total sugars, biomass, pigments, pH and moisture content were analysed. Results: Initial moisture content of 75% and 1g.L-1 of nitrogen were determined as optimal for pigment production after optimization studies. The highest pigment concentration was reached in 8 days of fermentation for red pigments, absorbance of 5.4 for the 500 nm range and in 4 days for yellow pigments absorbance of 8.7 for 410 nm. The maximal growth rate (13.37 g/100 g of protein) was reached at 8 days of fermentation coinciding with the highest red pigment concentration. Antimicrobial activity of the pigment produced were performed. Conclusion: It was observed that the association of cassava by-products are an alternative for the production of red and yellow pigments of the fungus Monascus purpureus NRRL1596.

Nutrition regulation for the production of Monascus red and yellow pigment with submerged fermentation by Monascus purpureus

Red pigment is one of natural coloring agents produced from the secondary metabolites of Monascus purpureus. Measures are taken to increase the pigment production. Effect of the extremely low-frequency magnetic field on the red and yellow pigment production of M. purpureus in liquid-state fermentation was estimated by exposing fermentation broth, medium and sterile distilled water used to prepare medium. The maximum yield of red and yellow pigment production had about 20 and 36 % increase for 8 days at 0.4 mT when treating fermentation broth compared to the control value. For sterile distilled water, a significant increase occurred at 0.4 mT compared to 0.1 mT for 5 h. However, the red and yellow pigment production decreased significantly at magnetic fields of 0.9 and 1.2 mT when treating fermentation broth and sterile distilled water. In contrast, the yield of both red and yellow pigment production decreased significantly compared with the control exposure for 3 and 5 h at a magnetic field of 0.4 mT. Different solution (cellular suspension, medium and sterile distilled water) exposure to magnetic fields has different bioeffect on M. purpureus SKY219. The appropriate magnetic field treatment could increase the efficiency of red and yellow pigment production.

Natural red pigments have been widely used as food and cosmetics additives. However, due to toxic byproducts or allergen issues, it is still necessary to look for some other red pigment products. This study proposed combinatorial strategies to improve production of a new kind of red pigments from the fungus Geomyces WNF-15A, isolated from Antarctica. A high-production medium was developed by statistical experimental design, which was further simplified for industrial use by single-factor experiments. Strain breeding by atmospheric room temperature plasma mutagenesis generated a mutant, Geomyces sp. WNF-15A-M210, which increased production of red pigments by 24.4% and shortened culture phase by 33.3% comparing with the wild-type. The production of red pigments by this mutant favored a weak alkaline condition but required only mild dissolved oxygen tension. Control of initial pH 8.5 (process pH around 7.5) increased red pigments production by 19% comparing with natural condition. Precursor and inhibitor addition experiments indicated that the red pigments were synthesized by polyketide pathway, and feeding 6mmol/L precursor of sodium acetate by three aliquots at days 3 to 5 improved biosynthesis of red pigments by 27%. Finally, the developed culture process was verified in a 5-L stirred tank bioreactor. The red pigments production of the pH regulation group reached 1.11-fold of the control and 1.95-fold of the precursor regulation group, respectively. This study provides high-production strain, optimized medium, and bioprocess for the possible industrial production of Antarctic Geomyces red pigments in future. PRACTICAL APPLICATION: Antarctic Geomyces red pigments showed high color value, nontoxic characteristic, and good water solubility. It holds potential for industrial use and is under development for food additive in China currently. This study provides an optional manufacturing process for this new kind of red pigments.

Since aeration has a crucial role in mass and heat transfer during solid-state fermentation (SSF), the aim of the present work was to find out how it related to biomass and red pigment production of Monascus purpureus TISTR3615 during SSF. The fungal consumption of oxygen, one of the air's constituents important for fungal metabolism, was used to explain the relationship between the aeration rate, the fungal growth, and the red pigment production during the SSF of rice. Findings showed that at increased aeration rates, the fungus absorbed more oxygen, which boosted fungal growth but decreased red pigment production. Conversely, red pigment production increased when the fungus' oxygen consumption decreased. Regression equations with an acceptable R2 were used to explain the relationship between M. purpureus' red pigment production and its oxygen consumption and aeration rate. The ideal oxygen concentration for increasing M. purpureus' capacity to produce red pigment was 0.0685 mmol/day/g of dry biomass, which produced red pigment at around 2,897 AU500nm/g of dry biomass. However, the optimal oxygen consumption of the fungus for overall red pigment production (including growth impact) was 1.390 mmol/day/g of dry biomass, which produced red pigment at around 2,787 AU500nm/g of dry fermented rice. The results of the present work could potentially be utilised in developing M. purpureus' red pigment production approach.

Monascus purpureus is a key fungus used to produce food pigments from secondary metabolites. Low-frequency magnetic field (LF-MF) affects production of M. purpureus secondary metabolites as one of the cultivation environments. The objective of this study was to measure optimum strength of induced magnetic field and duration of exposure for yellow and red pigment production of M. purpureus in solid-state fermentation. There was a 65.4 and 59.2 % increase in peak yield of yellow and red pigments compared to the control when the treatments were exposed to a magnetic field induction at 0.4 mT on the 8–9th day of incubation time. From kinetics of yellow and red pigment production, yield of yellow and red pigments was significantly increased in the samples exposed to magnetic field from the 8th to 11th day compared with that of the control group. LF-MF could increase the production rates of yellow pigments from 221.25 to 381.75 and red pigments from 165.00 to 292.50 U/g day. It could also significantly prolong the quickly generate time of yellow pigment. The appropriate magnetic field treatment could increase the efficiency of yellow and red pigment production.

Solid-state fermentation for the production of Monascus pigments from soybean meals

The used of synthetic dyes have various negative effects on human health. Roomates pigment produced by Monascus purpureus mold can be used as an alternative natural food coloring. The research on the effect of inoculum concentration’s M. purpureus to pigment production on the jackfruit seed flour has been done. The objective of research to is to investigate the effect of inoculum concentration’s M. purpureus to the production of red, yellow and orange pigment on the jackfruit seed flour. The concentrations used were 0%, 5%, 10%, and 15% (v/w). The result of the data analysed using One-Way ANOVA showed that the inoculum concentration affected the production of red pigment M. purpureus, as well as the data analysis using the Kruskal-Wallis showed that inoculum concentration has influence on the production of yellow and orange pigments. Inoculum concentration of 15% is the optimum concentration for the production of red, yellow and orange pigments with 0:10, 0:50 and 0:20 absorbance units per gram of sample respectively. Based on the results of the research, it can be concluded that inoculum concentration of M. purpureus influenced the production of red, yellow and orange pigments.

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

Factors such as environmental conditions and nutrients are significant for successful growth and reproduction of microorganisms. Manipulations of the factors are the most effective way to stimulate the growth of the microorganism, which can be used to optimize the yield of a product. In this study, Central Composite Design (CCD) of Response Surface Methodology (RSM) was used to optimize the production of red pigment by Monascus purpureus FTC 5356 using the petioles of oil palm fronds (OPF) as a substrate in solid state fermentation (SSF). The data was analyzed using Design Expert Software. The optimum combination predicted via RSM was confirmed through experimental work. The interactions between three variables such as initial moisture content (%), initial pH value (pH), and peptone concentration (%) were studied and modelled. The statistical analysis of the results showed that the optimal conditions for red pigment production 47 AU/g with the biomass of 425.1 mg/g was at 55% initial moisture content, 3% of peptone, and at pH 3. The RSM results showed that the initial pH value had a significant effect on red pigment production (P-value &lt;0.05). The validation of these results was also conducted by fermentation with predicted conditions and it was found that there was a discrepancy of 0.39% between the values of the experimental result and those of the predicted values. ABSTRAK: Keadaan persekitaran dan nutrien merupakan faktor-faktor penting dalam pertumbuhan mikroorganisma. Manipulasi faktor-faktor tersebut adalah kaedah terbaik bagi meningkatkan pertumbuhan mikroorganisma dan mengoptimumkan penghasilan produk. Kajian ini mengguna pakai Rekaan Gabungan Pusat (CCD) melalui Kaedah Tindak balas Permukaan (RSM) bagi penghasilan pigmen merah optimum oleh Monascus purpureus FTC 5356 menggunakan batang pelepah kelapa sawit (OPF) sebagai perumah dalam proses penapaian pepejal (SSF). Data telah dianalisis menggunakan perisian Design Expert. Gabungan parameter optimum seperti cadangan RSM telah disahkan secara eksperimen. Interaksi antara tiga pemboleh ubah seperti kandungan lembapan awal (%), nilai pH awal (pH), dan kepekatan pepton (%) telah dikaji dan dimodelkan. Analisis statistik menunjukkan penghasilan optimal pigmen merah adalah pada 47 AU/g dengan biomas sebanyak 425.1 mg/g, pada 55% lembapan awal, 3% pepton dan pada pH 3. Hasil keputusan RSM menunjukkan pH awal memberikan kesan signifikan kepada penghasilan pigmen merah (nilai P &lt;0.05). Pengesahan analisis juga telah dijalankan melalui proses penapaian dan hasil ujikaji mendapati 0.39% lebih tinggi daripada nilai jangkaan.

By increasing the undesirable side effects of synthetic food pigments on human health, using safe natural food pigment become an urgent issue. Incorporate corn starch with oils conducted a high impact on red pigment production by Monascus purpureus. Fortification the medium with sesame oil raised the pigment production by 80% and the dry mass by 63% compared with free oil medium. Response surface methodology maximizes the production with 114.6% (12.8 A500) using medium constituents (g/l); Sesame oil 5; Corn starch 30; Yeast extract 1.5; KH2PO4 2.5 and MgSO4.7H2O 0.1. After evaluating red pigment stability in three common food components, citric acid showed a great effect on residual stability percentage compared with ascorbic and salicylic acid which decrease slightly the residual stability percentage at light and dark conditions. The mitotic index of red pigment was lower than the negative control at all tested concentrations. Different types of mitotic chromosomal abnormalities e.g. lagging chromosome, chromosomal bridge, chromosome and chromatin fragments, outside chromosome, chromosomal stickiness and micro nuclei were recorded. Insignificant increase in total mitotic aberrations percentage in all tested root tips treated with all concentrations of red pigment (1.23, 1.58, 1.63, 2.32 and 2.40%) compared with negative control (0.91%). There was a significant increase in total aberrations percentage after treatment with all concentrations (10, 15, 20 and 25%) of positive control (2.93, 3.00, 3.55 and 6.53 respectively) except (5%) which was insignificant (2.71%). From the previous data, this red pigment can be used as an alternative safe pigment in the food industry.