524 Chiang Mai J. Sci. 2014; 41(3)

Chiang Mai J. Sci. 2014; 41(3) : 524-530http://epg.science.cmu.ac.th/ejournal/Contributed Paper

Collective Effects of Stress on Optimization ofPigment Production by Monascus purpureusRashmi Dikshit and Padmavathi Tallapragada*Department of Microbiology, Centre for PG Studies, Jain University, 18/3, 9th Main road, 3rd Block, Jayanagar,Bangalore – 560011, Karnataka India.*Author for correspondence; e-mail: vam2010tpraviju@gmail.com; t.padmavathi@jainuniversity.ac.in

Received: 11 March 2013Accepted: 29 May 2013

ABSTRACTMonascus sp. is well-known for pigment production and its application as food

colorants. The aim of this study was to optimize the pigment yield and biomass producedfrom Monascus purpureus in submerged culture under the combined effect of various stressconditions. In this experiment, the thermal stress was induced by incubating the spores atvarious temperature ranges. For inducing osmotic stress, varied concentrations of NaCl,glycerol and peptone were used. Pigment and biomass was statistically optimized withresponse surface methodology (RSM). A 24 full factorial central composite design (CCD) wasapplied to elucidate the combined effects of above mentioned stress conditions. The resultsshowed that the linear effect of glycerol on pigment yield was more significant. The optimumcalculated values for the test variables in coded factors was estimated at 71.25 colour valueunits (CVU)/ml as against the experimented value of 68.67 CVU/ml at 70°C for temperature,1.087 mol/l (w/v) for glycerol, 1.25% (w/v) for peptone and 0.25% (w/v) for NaCl.Statistically optimized pigment yield was amazingly found to be almost two fold as comparedto the control.

Keywords: optimization, response surface methodology, thermal stress, Monascus sp.

1. INTRODUCTIONPigments are natural colorants synthesized

by natural sources like plants and micro-organisms. In food industry these pigmentsare used as additives, colour intensifiers andantioxidants, etc. For these reasons, many ofthese compounds have been produced,isolated, and characterized [1]. Out of these,the genus Monascus produces a wide range ofpigments and provides with a potentialopportunity for the researchers. Hence specialattention has been focused on the strains

belonging to the Monascus genus offilamentous fungi [2]. Monascus purpureus,a homothallic fungus which is classified tothe Ascomycetes family Monascaceae [3].Monascus strain has ability to producesecondary metabolites of polyketide structurewhich are synthesized by the polymerizationof acetyl and propionyl subunits [4]. Apartfrom producing red, orange and yellowpigments, these secondary metabolites alsocontain lovastatin (monocolin K) known to

Chiang Mai J. Sci. 2014; 41(3) 525

reduce blood lipid levels, γ-aminobutaric acidused for lowering blood pressure levels,dimerumic acid known as an antioxidant andmonascin as an anti-inflammatory agent [5].It is known that production of severalsecondary metabolites is triggered by stress.Under stress conditions, cell maintains energysources and essential metabolites, thuscreating conducive growth environment forconversion of primary metabolites intosecondary metabolites [6]. Response surfacemethodology (RSM) is a powerful statisticaltool. It has a key benefit over ‘one-factor-at atime’ approach as it allows the evaluation ofthe effect of multiple variables and theirinteractions on the output variables withreduced number of experimental trials [7].

Present study was carried out with theobjective to investigate collective effect ofdifferent stress conditions on the productionof red pigment in submerged culture byMonascus purpureus using RSM as a tool.

2. MATERIALS AND METHODS2.1 Microorganism

The culture, Monascus purpureus MTCC410 procured from the Microbial TypeCulture Collection, IMTECH, Chandigarh,India and was maintained on Potato DextroseAgar (PDA) medium and incubated at 30°Cfor 7 days [8].

2.2 Inoculum PreparationSpore suspension of Monascus purpureus

MTCC 410 was prepared with sterile distilledwater and diluted to a concentration of 5×105

spores/ml [9].

2.3 Submerged FermentationOptimization of red pigment and

biomass was investigated in potato dextrosebroth fungal media. 50 ml media wereprepared and autoclaved at 121°C for 20minutes. Medium pH was adjusted to 5.5.

After cooling, this media was inoculatedwith 0.5 ml of M. purpureus culture andincubated for 16 days in static condition[8]. The influence of osmotic stress wasinvestigated by adding glycerol at differentconcentrations namely 0.25, 0.5, 0.75, 1 and1.25 mol/l and peptone and NaCl atconcentrations namely 0.25, 0.5, 0.75, 1 and1.25% (w/v) to the media prior to autoclaving[6]. To investigate the effect of thermalstress on spores, the spore suspension wassubjected to different temperatures (30, 40,50, 60 and 70°C) for one minute prior toinoculation. These spores were used asinoculum [6].

2.4 Estimation of Pigment Yield and DryCell Weight

The biomass was separated from brothby filtration using pre-weighed filter paperwashed with distilled water and dried in anoven at 50°C. The results were expressed ing/l [10]. Filtrate was centrifuged at 10000×gfor 15 minutes and pigment concentrationwas estimated using colorimeter at 510 nm.The absorbance values were converted intopigment units using by the following formula:

Colour value = O.D. × dilution × volume ofextracts / Amount of sample (ml) [11].

2.5 Experimental DesignExperiment was designed according to

Central Composite Design (CCD) of RSMusing MATLAB® software package Version7.5.0.342 (R2007b) from The Math Works,Inc. for selected four stress condition viz.spores treated at different temperatures,different concentrations of glycerol, NaCland peptone (Table 1). The experimental andpredicted values for pigment yield andbiomass were calculated as per the secondorder quadratic model using CCD andpresented in Table 2 [12].

526 Chiang Mai J. Sci. 2014; 41(3)

Table 1. Process variables and their levels.Factor

Temperature [°C]Glycerol [mol/l]Peptone [%]NaCl [%]

Code

X1X2X3X4

Actual factor level at coded factor levels of-230

0.250.250.25

-1400.50.50.5

050

0.750.750.75

160111

270

1.251.251.25

Table 2. Central composite design (CCD) of factors in coded levels with biomass andpigment yield as response.

All experiments were carried in triplicates; the above mentioned values are the mean values

RunNo.

1.2.3.4.5.6.7.8.9.10.11.12.13.14.15.16.17.18.19.20.21.22.23.24.25.26.27.28.29.30.

Temp[°C]

404040404040404060606060606060605050505050503070505050505050

Glycerol[mol/l]

0.51

0.51

0.51

0.51

0.51

0.51

0.51

0.51

0.251.250.750.750.750.750.750.750.750.750.750.750.750.75

Peptone[%]

0.50.511

0.50.511

0.50.511

0.50.511

0.750.750.251.250.750.750.750.750.750.750.750.750.750.75

NaCl[%]

0.50.50.50.51111

0.50.50.50.51111

0.750.750.750.750.251.250.750.750.750.750.750.750.750.75

Biomass[g/l]

Experimental10.221.48.618.68.018.65.617.10.714.88.020.811.616.611.819.29.029.28.411.410.211.810.012.617.019.216.018.217.218.0

Predicted9.620819.87506.775019.80428.141717.57085.370817.57508.741715.42089.420818.875010.787516.641711.541720.170810.354229.237510.254210.937511.787511.604211.137512.8542

17.617.617.617.617.617.6

Pigment Yield[CVU/ml]

Experimental23.756.05

31.87542.521.2516.62519.37532.5

28.1257.2538.7537.516.2515.521.2541.2515.515.8816.2526.2533.12517.530.122.521.521.8922.820.522.121.89

Predicted27.10199.656933.268537.436020.185216.977716.176934.581926.10607.586035.535238.627718.451914.169417.706035.036017.147117.032112.132939.166231.966221.457927.970427.428721.7821.7821.7821.7821.7821.78

Chiang Mai J. Sci. 2014; 41(3) 527

3. RESULTS AND DISCUSSIONHeat treated spores were examined under

phase contrast microscopy (4×). There wasno significant difference seen in sporemorphology when the spores were treatedwith 40°C and without heat treatment(Figure 1a). However the spores treated at70°C has shown appreciable difference(Figure 1b). Cleistothesium was rupturedwhen the Monascus culture was treated at70°C and these pigmented spores werespread out from the fruiting body. It can be

due to the fact that at extreme temperaturecytoplasm losses water to the surroundingmedium and results in the overproduction ofthe hydrophobic pigment. It was reported thatthe walls of Monascus cells suffer fromhydrolysis, and the Monascus cells tends tooverproduce hydrophobic substances such aspigments for blocking these enzymatic attacks[13]. Shea and Walsh [14] concluded thatunderstanding of cell morphology could bea key to enhance and optimize product activity.

Figure 1. Phase contrast microscopic pictures showing heat treated spores a) at 40oC andb) at 70oC.

(a)

(b)

3.1 Optimization of Red Pigment YieldThe equation explaining the relationship

of the four variables can be written as

.........(1)

Where X1 is the temperature variable, X2 isthe glycerol concentration, X3 is the

peptone concentration and X4 is the NaClconcentration. Substituting the values for theconstants, the equation for calculating thepigment yield takes the following form

Pigment yield ( at 510 nm) = 137.1053 -1.8982X1 - 73.0725X2 -50.0717X3 - 47.8633X4

- 0.1075X1X2 - 0.3262X1X3 - 0.0738X1X4 +86.45X2X3 + 56.95X2X4 - 40.7X3X4 + 0.0148X1

2

- 18.7617X22 + 15.4783X3

2 + 19.7283X42 ....(2)

21

22 3

242

Y = b0+ b1x1 + b2x2 + b3x3 + b4x4 + b5x1x2 +b6x1x3 + b7x1x4 + b8x2x3 + b9x2x4 + b10x3x4 +b11x + b12x + b13x + b14x

528 Chiang Mai J. Sci. 2014; 41(3)

A grid of 40 points each for the fourvariables was generated. The interactioneffects of variables on pigment yield werestudied by plotting 3D surface curvesagainst any two independent variables,while keeping another variable at its central(0) level. The 3D plots of the calculatedresponse and contour plots from theinteractions between the variables (1600data points) are shown in Figure 2.

From the results it is evident that forthe interaction between spores treated withdifferent temperature and glycerol, thepigment yield was found to be higher atextreme temperatures, either high or low andat middle range of glycerol concentration(0.698 mol/l at 70°C and 0.8 mol/l at 40°C).There was a decrease in the pigmentyield observed at mid temperature rangeirrespective of the variation in the glycerolconcentration. The maximum value ofpigment yield was about 28.03 CVU/ml(Figure 2a). It was reported that, manyorganisms when exposed to elevatedtemperatures, rapidly synthesize a highlyconserved set of protein called heat shockprotein, these proteins provide an

adaptation to the organisms to survive insuch hypothermic stress condition [15, 6].

The interaction effect of peptone andglycerol on pigment yield showed extremetrends. The area for the higher pigment yieldwas intensified on the higher side of theconcentration for these variables. However,for lower peptone concentration, thepigment yield showed a decreasing trendwith increasing glycerol concentration andreached to theoretically negative values. Thisshows that synthesis of red pigment is quitedependent on C-N ratio. The maximumpigment yield of 34.2 CVU/ml was obtainedat 1.25% (w/v) peptone concentration and1.25 mol/l (w/v) glycerol concentration(Figure 2b). It can be concluded that thepeptone concentration was the most signicantvariable for pigment production in our study.Formation of red pigment is stronglyregulated by the amino acid used as nitrogensource. It is reported that amino acid acts asside chain precursors for the production ofwater soluble red pigment [16]. The additionof glutamate resulted in an increase inMonascus pigment yield, but only combinedwith high peptone concentrations [17].

Figure 2. 3D surface curves and contour plots showing the interaction effects of dominantvariables on pigment yield (CVU/ml). a) glycerol vs. temperature and b) peptone vs.glycerol.

Chiang Mai J. Sci. 2014; 41(3) 529

3.2 Impact of Stress on BiomassThe equation explaining the

relationship of the four variables onbiomass works out to be

Biomass (g/l) = -53.8594 + 1.4635X1 +21.3083X2 + 13.0083X3 + 16.3417X4 -0.3575X1X2 + 0.3525X1X3 + 0.3525X1X4 +11.1X2X3 - 3.3X2X4 + 0.3X3X4 - 0.014X1

2 +8.8733X2

2 - 28.0167X32 - 23.6167X4

2 .........(3)

The highest value of the biomass wasabout 29.57 g/l at a temperature of 55.17°Cand 1.25 mol/l glycerol concentration. Similareffect was seen with NaCl and glycerol.The productive range of NaCl concentrationshowed a symmetrical divergence from0.66-0.9% (w/v) at glycerol concentrationof 0.25 mol/l to 0.28 - 1.1 % (w/v) at 1.25mol/l. With peptone and glycerol, thebiomass increased with increase in glycerolconcentration but at mid peptoneconcentration .

Under conditions where temperatures

elevated to the normal growth range, cellsexperience stress due to the damaging effectof heat on intracellular macromolecules, suchas heat-sensitive enzymes, and to cellmembranes [18]. It has been reported that,both prokaryotic and eukaryotic cells areable to respond on sub-lethal temperatures(heat shock) by undergoing rapid and massivesynthesis of a subset of cellular proteinscommonly referred to as heat shock proteins(HSP) [19].

The analysis of variance, for the redpigment production and biomass wascalculated by coefficient of determination (R2)(Table 3). R2 value (goodness of fit model),was 0.8956 for red pigment yield, whichindicated that 89.56% of the total variationin the observed response value could beexplained by the model. For biomass R2 wasestimated at 0.9558 (accuracy of 95.58%)It was also observed that the model was highlysignificant with a p-value of 5.6 × 10-5forpigment yield and a p-value of 10-7 forbiomass.

4. CONCLUSIONSThe second order regression Eq. (2)

which was obtained from CCD was solvedusing MATLAB software. The optimumvalues for the test variables in coded factorswere found to be 70°C for temperature, 1.08mol/l % (w/v) for glycerol, 1.25% (w/v) forpeptone and 0.25% (w/v) for NaCl for thepredicted maximum red pigment yield of71.25 CVU/ml. Experiment was carried outwith the above predicted optimum conditionsin submerged culture and the pigment yieldof 68.67 CVU/ml was obtained. The

closeness of the predicted value with theexperimental value is the measure of thefittingness of the proposed model and thussubstantiating the model. These results can beexploited at industrial level to scale up thepigment yield and the product can be used asa potential food colorant.

REFERENCES[1] Alejandro M., Catalina P., Julio C. M.,

Gabriela M. and Crist bal N. A., Redpigment production by Penicilliumpurpurogenum GH2 is influenced by

Table 3. Analysis of variance (ANOVA) for response surface quadratic model.

SSE

258.7825

35.7842

Degree of freedom

14

14

f-value

9.1936

23.1479

p-value

0.000056

0.0000001

Mean Square

17.2522

2.3856

R2

0.8956

0.9558

Adj. R2

0.7982

0.9145

For Pigment yield

For Biomass

530 Chiang Mai J. Sci. 2014; 41(3)

pH and temperature, J. Zhejiang Univ.Sci. B (Biomed. & Biotechnol.), 2011;12(12): 961-968.

[2] Blanc P.J., Laussac J.P., Bars J.L., BarsP.L., Lorret M.O., Pareilleu A., PromeD., Prome J.C., Santene A.L. and GomaG., Characterisation of Monascidin-afrom Monascus as citrinin, Int. J. FoodMicrobiol., 1995; 27: 201-213.

[3] Pattanagul P., Pinthong R.,Phianmongkhol A. and LeksawasdiN., Review of Angkak production(Monascus purpureus), Chiang Mai J.Sci., 2007; 34: 319-328.

[4] Pongrawee N. and Lumyong S.,Improving solid-state fermentation ofMonascus purpureus on agriculturalproducts for pigment production, FoodBioprocess Technol., 2011; 4: 1384-1390.

[5] Panda B.P. and Ali M., Reduction ofcitrinin biosynthesis by fatty acids inMonascus fermented food, WorldMycotoxin J., 2012; 5(2): 163-168.

[6] Sumathy B., Carlos R.S. and PandeyA., Effect of stress on growth, pigmentproduction and morphology ofMonascus sp. in solid cultures, J. BasicMicrobiol., 2007; 47: 118-126.

[7] Liyana P.C. and Shahidi F.,Optimization of extraction of phenoliccompounds from wheat using surfaceresponse methodology, Food Chem.,2005; 93: 47-56.

[8] Dikshit R. and Padmavathi T.,Comparative study of Monascussanguineus and Monascus purpureus aspotential sources for red pigmentproduction. Int. J. Pharm. Biol. Sci.,2012; 3(4): 885-895.

[9] Md. Makhmur Ahmad, M., NomaniS. and Bibhu B.P., Panda screening ofnutrient parameters for red pigmentproduction by Monascus purpureusMTCC 369 under submerged fermen-tation using Plackett-Burman Design,Chiang Mai J. Sci., 2009; 36(1): 104-109.

[10] Mukherjee G. and Singh S.K.,Purication and characterization of anew red pigment from Monascuspurpureus in submerged fermentation,Process Biochem., 2010; 46: 188-192.

[11] Ratana S. and Toshima Y., Solid-statefermentation for yellow pigmentsproduction by Monascus purpureus,World j. Microbiol. Biotechnol., 1987;6: 347-352.

[12] Subhagar S., Aravindan R. andViruthagiri T., Statistical optimizationof anticholesterolemic drug lovastatinproduction by the red mold Monascuspurpureus, Food Bioprod. Process.,2010; 88: 266-276.

[13] Shin C.S., Kim H. J., Kim M. J. andJu J.Y., Morphological change andenhanced pigment production ofMonascus when co-cultured withSaccharomyces cerevisiae or Aspergillusoryzae. Biotechnol. Bioeng., 1998; 59:576-581.

[14] O’Shea D.G. and Walsh P.K.,Morphological characterization of thedimorphic yeast Kluyveromyces marxianusvar. marxianus NRRLy2415 by semiautomated image analysis, Biotechnol.Bioeng., 1996; 51: 679-690.

[15] Schlesinger M.J., Heat shock protein,J. Biol.Chem., 1990; 265: 12111-12114.

[16] Lin T.F. and Demain A.L., Leucineinterference in the production of watersoluble red Monascus pigments, Arch.Microbiol., 1994; 162: 114-119.

[17] Silveira S.T., Daniel J.D. and AdrianoB., Pigment production by Monascuspurpureus in grape waste using factorialdesign, LWT- Food Sci. Technol., 2007;41: 170-174.

[18] Henle K.J., Nagle W.A., Moss A.J.and Herman T.S., Polyhydroxycompounds and thermo tolerance: Aproposed concatenation, Radiat. Res.,1982; 92: 445-451.

[19] Lindquist S., The heat shock response,Annu. Rev. Biochem., 1986; 55: 1151-1191.