Stability of Colorants from Ultrasonic Induced Monascus ...ipcbee.com/vol46/026-ICBEC2012-G20014.pdf · Stability of Colorants from Ultrasonic Induced Monascus purpureus Issara Wongjewboot,

Stability of Colorants from Ultrasonic Induced Monascus purpureus

Issara Wongjewboot, Touchchakorn Bowdang and Sasithorn Kongruang+

Department of Biotechnology, Faculty of Applied Science, King Mongkut’s University of North Bangkok, Bangkok, Thailand 1518 Piboonsongkram Road, Bangsue, Bangkok, Thailand 10800

Abstract. Natural colorants derived from polyketide compounds of Monascus purpureus have been extensively used in pharmaceuticals and foods to replace the synthetic colorants. A ultrasonic treatment at 45 kHz for 2min at 28 0C for induced mutation was applied to Thai Isolated Monascus purpureus TISTR 3385 fermented in a modified yeast extract supplemented with cassava starch and monosodium glutamate to generate extracellular pigment Monascus mutant producers. Four generations of ultrasonic induced were obtained as G1-G4 and wild type, G0 and the derived extracellular pigments were tested for the ranges of pH and solvent stability. Results showed that yellow pigments of both mutants and wild type showed higher stability on pHs and solvents than other pigments especially at the basic pH values (> 7.0-13.0). When compared with the wide ranges of solvent stability, wild type showed significantly higher stability over other generations at which hexane was the most suitable and the yellow pigments of all generation were the best sustainable colorant. These results indicate that Monascus yellow pigments of all generations may possibly be used as colorants in foods having neutral, slightly acidic and basic pH values.

Keywords: Monascus purpureus, Biopigment, Color stability, Mutation, Citrinin, Ultrasound

1. Introduction Monascus can produce at least eight major pigments as called monascin and ankaflavin for yellow,

monascorubrin and rubropunctatin for orange and monascorubramine and rubropunctamine for red pigments. The comparison of pharmaceutical ability of these pigments from Shi and Pan, 2011 [1] report showed that the red mold rice and red mold adlay, the red mold dioscorea had high levels of anti-inflammatory yellow pigments, monascin and ankaflavin. Recently, Compoy et al. [2] reported that Monascus species can also produce two new pigments, a red pigment and a yellow pigment as shown in figure 1, that they characterized by nuclear magnetic resonance. Other functional metabolite compounds such as isoflavones, polyketide monacolins, citrinin, γ- aminobutyric acid and dimerumic acid have also proven to be an alternative approach and a beneficial effect to use as active compounds in pharmaceutical products such as in cholesterol lowering drug, blood pressure control, anticancer, anti-fatigue and Alzheimer’s disease management [3-5]. Although there are many hyperpigment strains, the stability over a wide ranges of conditions still need to prove for an applicable utilization in industries. For the useful Thai isolated Monascus both wild type and mutant strains at which their cultivation, structures, functions , the stability over the ranges of pH and solvent and antibiotic application have studied and reported [6-11]. The unstability in foods and pharmaceutical application for extreme pH, solvent, heat and light cause the consideration for seeking for the screening approach to find appropriate strains without citrinin coproduction. Natural colorants are normally easy to degrade the exposure to different pHs and solvents, resulting in a loss of appearance acceptability.

In this paper, we study the stability of derivatives of monascus pigments obtained from ultrasonic induced mutation of wild Thai isolated types of M. purpureus TISTR 3385.The extracellular pigments of both wild type (G0) and mutants (generations 1-4, represented as G1-G4) obtained by fermentation of a

+ Corresponding author. Tel.: + 662-913-2500 ext.4303; fax: +662-5878259. E-mail address: [email protected].

2012 3rd International Conference on Biology, Environment and Chemistry IPCBEE vol.46 (2012) © (2012) IACSIT Press, Singapore

DOI: 10.7763/IPCBEE. 2012. V46. 26

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modified yeast extract supplemented with cassava starch and monosodium glutamate were evaluated for pHs, temperatures and solvent stability.

(A) (B)

Fig. 1: Chemical structures from mass spectrometry and NMR spectroscopy identification of (A) red pigment, 9-hexanoyl-3-(2-hydroxypropyl)-6a-methyl-9,9a-dihydro-6H-furo[2,3-h]isochromene-6,8(6aH)-dione and (B) yellow

pigment, found by Compoy et al.[2006].

2. Materials and Methods

2.1. Reagents Culture media (potato dextrose agar, yeast extract powder, peptone, and malt extract powder) were

purchased from Himedia Laboratories Pvt. Ltd., India. N-source-medium (monosodium glutamate) and C-source as cassava starch were purchased from commercially available in Thailand. L-glucose was a product of Sigma-Aldrich Co. Solvents as hexane, ethanol, propanol, methanol, ethylether and double distilled water were obtained from Labscan, Thailand. Citrinin standard was purchased from Sigma Chemical Company.

2.2. Strains and Culturing Conditions All Monascus purpureus TISTR 3385 was obtained from Thailand Institute of Scientific and

Technological Research, Thailand. The stock of culture in freeze-dried ampoules was activated in YM broth at 30°C for 2 days and transferred to PDA and further incubated for 7 days. The radial growth of each of the four generations was measured in duplicate by point inoculation at the center of 8 PDA petridish. The cultures were then incubated at 30°C for 14 days. The radius of each colony was measured from the center of the dish along 2 perpendicular axes at intervals of 2 days over a period of 14 days. The growth rate of each generation was calculated and reported by using linear regression.

2.3. Cultivation for Pigment Production The stock of culture in freeze-dried ampoules was activated in YM broth at 30°C for 2 days and

transferred to PDA and further incubated for 7 days. A spore suspension of 106 spores/ml of 0.1% Tween 80 solvent was initially transferred to a 30 ml autoclaved liquid medium of modified YM broth (5% peptone, 3% yeast extract powder, 3% monosodium glutamate, 10% cassava starch in distilled water). An Erlenmeyer flask containing 30 ml of seed culture medium was then incubated in a shaking incubator at 200 rpm, 300C for 7 days. The culture was then transferred into a flask containing 300 ml of the same medium. The culture media was then separated by centrifugation at 4°C for 20 min (10,000 ×g). The mycelia were extracted and filtered again through a 0.45-μm membrane. The supernatant was kept for further analysis.

2.4. Ultrasonic Induced Mutation Treatment The culture medium of seed culture of wild type M. purpureus TISTR 3385 was filtrated and the seed

suspensions in 7% saline solution were then treated by ultrasound at 45 kHz for 2 min at 28°C. After the ultrasound, the mycelia were washed with double distilled water. They were then used as a seed for radial growth determination and for cultivation for pigment production by the procedures described above. The mycelium grown in the PDA was used as generation 1 (G1). The ultrasonic treatment was repeated on G1 to obtain a second generation (G2). The ultrasound treatment was repeated to obtain generations G3 and G4.

2.5. Pigments, Citrinin and Monacolin K Determination Pigment concentrations were Pigment concentrations were estimated using spectrophotometer (UV-vis

spectrometer, Shimadzu Co., UV-1201, Tokyo) at 340, 440 and 480 nm for yellow, orange and red pigments.

123

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124

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125

3.3. Effect of Solvent on Pigment Stability Several solvents were used to test the extracellular pigment of all generations as illustrated in fig.4 (a-e).

Results showed that all generations were sustained in ethanol and mostly retained the stability between 20-90%. The wild type showed significantly higher solvent stability over other generations at which hexane was the most suitable solvent to maintain colour. Propanol and distilled water were not appropriate to use as solvents for the pigments of TISTR 3385 strain both wild type and mutants. For solvent stability, yellow pigments of all generation were the best sustainable colorant.

3.4. Citrinin and Monacolin K Determination The examination of the chromatograms for the pigments obtained from the four M. purpureus strains and

could not detect Monacolin K in all generations. Citrinin peaks were found in some generations however it were lower than a standard safety level. In conclusion, we have reported that Monascus pigments of all generations of TISTR 3385 are more stable under basic conditions. Overall, yellow pigment of all generations showed a great possibility to use as a robust colorant for food additive and a tendency to apply in textile industry.

4. Acknowledgements

The authors are grateful to financial support by The Thailand Research Fund under grant number 2552A11902010.

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[3] Y.C. Shi and T.M., Pan Beneficial effects of Monascus purpureus NTU 568-fermented products: a review. Appl Microbiol Biotechnol .2011, 90:1207–1217.

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