Effect of Amino Acids on Pigments, Citrinin, and Lovastatin Production by Monascus Purpureus Under Static Conditions

BIOLOGIJA. 2017. Vol. 63. No. 2. P. 160–168 © Lietuvos mokslų akademija, 2017 Effect of amino acids on pigments, citrinin, and lovastatin production by Monascus purpureus under static conditions Padmavathi Tallapragada*, Monascus spp. is known to produce many secondary metabolites including pigments, statin, and undesired mycotoxin. Rashmi Dikshit, M. purpureus MTCC 410 strain was grown statically for ten days in potato dextrose broth supplemented with 1% UV steri- Mudaheranwa Phocas, lized amino acids. Whatmann filter No. 1 was used to separate the developed mycelia from the broth to get the intracellular pig- Madhusudan. M.R., ment as the extracellular one is left in the filtrate. The pigment concentration was estimated by the calorimetric method for dif- Sanjana Samprathi ferent wavelengths and expressed in colour value units (CVU). The presence of citrinin in the growth medium was checked un- Department of Microbiology, der UV light at 350 nm and quantification was done with high- Centre of PG Studies, Jain University, 18/3, 9th Main, Jayanagar 3rd Block, performance liquid chromatography column along with loop in- Bangalore, India jector of 20 μl, and Shimadzu CLASS-VP version 5.032 software. The maximum biomass (143.6 g/l) was observed with supplementation of 1% D-serine to the medium, whereas the maximum intracellular pigment yield was observed with supplementation of L-histidine monohydrochoride (yellow – 4.48, orange – 3.97 and red pigment – 2.0 CVU/ml). The maximum extracellular pigment yield was observed with supplementation of glycine (yellow – 2.18, orange – 1.65 and red pigment – 1.38 CVU/ml to the growth medium). The maximum lovastatin yield was observed with supplementation of L-cysteine mono hydrochloride and concentration of 2064 mg/l. Maximum citrinin (1.29 mg/l) was observed with supplementation of DL-norleucine to the growht medium. M. purpureus requires suitable concentration of organic nitro- gen in the form of amino acids for a higher yield of secondary metabolites such as supplementation of 1% L-cysteine monohydro- chloride or L-tyrosine in the growth medium under submerged cultivation. None of the tested amino acids produced citrinin under experimental conditions making the outcome beneficial for industrial purposes. Keywords: extracellular pigment, citrinin, amino acids, Monas- cus purpureus, biomass * Corresponding author. E-mail: [email protected], [email protected] Effect of amino acids on pigments, citrinin, and lovastatin production by Monascus purpureus under... 161 INTRODUCTION Lovastatin works as another cholesterol-low- ering agent by suppressing cholesterol synthesis Monascus purpureus is a mould species, pur- via inhibition of HMG-CoA reductase enzyme, plish-red in colour, in Ascomycota division, which mediates the biosynthesis of cholesterol in Monascaceae family. For a long time it has been liver and is known as a potent inhibitor simvas- traditionally important in China, Thailand, and tatin (Ahmad et al., 2009). Thus the presence of Japan for production of red rice wine, red soy- lovastatin in bioproducts of an organism is an ad- beans, cheese, and ang-kak rice. It was found ca- ditional advantage making Monascus spp. impor- pable of producing safe natural pigments along tant in medical and pharmaceutical industries by with other therapeutically important secondary cooperatively working with other compounds or metabolites, including citrinin and statins (Du- individually to stimulate body cells in the treat- fosse et al., 2005). ment of Alzheimer’s disease or cancer, induction It contributes to consumers’ favourite food of apoptotic cell death, reduction of inflamma- colours, from orange-red to violet-red, by pro- tion, inhibition of tumorigenesis and viral repli- ducing at least six major related pigments pre- cation (Nicoleta et al., 2012). ferred over other synthetic pigments nowadays The present study was conducted to screen used in food industry, and are categorized into amino acids for biomass, pigment, and citrinin three main groups: yellow pigments (monas- and lovastatin production under static condi- cin and ankaflavin), orange pigments (rubro- tions. punctain and monascorubin), and red pigments (rubropunctamine and monoscorubramine) MATERIALS AND METHODS (Wang et al., 2007). They are synthesized as water insoluble, aminophiles and unstable in Culture extreme pH in the cytosol from acetyl-coA Monascus purpureus MTCC 410, obtained from through multi-enzymatic polyketide synthase IMTECH, Chandigarh India, was maintained complex (Balakrishnan et al., 2014) becoming on Potato Dextrose Agar (PDA) medium at 30°C water soluble after being in contact with present amino acids. Their aminophile character- for seven days and preserved at 4°C, with sub- istic makes them to be associated with cellular culturing once every four weeks; its purity was proteins or with the cell wall, and this makes checked regularly (Dikshit, Tallapragada, 2013). them difficult to be extracted. Therefore their extraction requires cell breakage and dissolu- Submerged fermentation tion in an organic solvent like methanol (Patri- Potato dextrose broth was sterilized by autoclav- cia et al., 2005; Liu et al., 2014). ing, supplemented with UV sterilized 1% amino However, as an undesired product, hepato- acids respectively, inoculated with a loopful of nephrotoxin citrinin is known to be produced M. purpureus, and incubated for ten days under along with pigments and it limits the use of static conditions (Dikshit, Tallapragada, 2013). Monascus as a producer of natural food colour- ants (Jongrungruangchok et al., 2004). Its pro- Biomass estimation duction is the main concern worldwide and After incubation, the mycelia was separated it has to be prevented using any possible tech- from the broth using Whatmann filter No. 1, niques like the use of mutagenic agents induc- obtained biomass was then weighed to deter- ing mutation in a specific gene of an organism mine the fresh biomass in grams per litre (Dik- or inhibition of a specific responsible polyketide shit, Tallapragada, 2013). enzymes (PKSs) (Kang et al., 2014). Supplemen- tation of specific amino acid and cultivation un- Pigment estimation der controlled conditions can enhance the pig- Ten millilitres of both extracts were taken in two ment production and inhibit citrinin production separate conical flasks and extra 10 ml of meth- (Sameer et al., 2005; Liu et al., 2005). anol was added and incubated in the shaker 162 Padmavathi Tallapragada, Rashmi Dikshit, Mudaheranwa Phocas, Madhusudan. M.R., Sanjana Samprathi incubatorfor 45 minutes, the filtrate was centri- 10 ml of 70% perchloric acid was added and fuged at 10,000 rpm for 10 minutes. The pigment heated until the iron dissolved. As iron dis- concentration was then determined by the col- solves very rapidly when the acid is hot, the re- orimetric method where absorbance was meas- action may become vigorous and the content ured at 450, 470, and 540 nm for yellow, orange, was cooled in the fuming hood. Then 40 ml of and red pigments respectively, and the colour this stock was mixed with 12 ml of 70% per- value units were calculated. Colour value = O.D. chloric acid and the volume was made up to x dilution x volume of extract/amount of sample 100 ml with ethanol to make Ferric perchlorate (ml) (Ratana, Toshima, 1987). reagent (Mohie et al., 2010). Lovastatin was estimated by different ali- Citrinin analysis quots of standard lovastatin (6 mg/ml) ranging The extract was transferred into vials and from 20 μl to 100 μl transferred into a series saturated until the volume reduced to half of of test tubes: 1 ml of alkaline hydroxylamine the original, 10 microliters of it was applied to reagent was added to each test tube, then pH was Silica gel 60 F254 Aluminium sheets (Merck, adjusted to 1.2 ± 0.2 by adding an appropriate Germany) using Ethyl acetate: acetone: wa- volume of 2 M HCl, followed by addition of 2 ml ter (4: 4: 1 v/v) as the mobile phase, and dried of ferric perchlorate reagent. After 25 minutes of plates were examined under UV light at 350 nm incubation at room temperature, the absorbance to observe fluorescent yellow bands indicating values of the red colour product formed were the presence of citrinin. For its confirmation, read at 510 nm (Mohie et al., 2010). The same High Performance Liquid Chromatography procedure was followed for the test samples. (HPLC) analysis was carried out, column with C18 with 250 × 4.6 mm width ID Lichrosper RESULTS AND DISCUSSION 100 and 5 μm particle size, along with loop in- jector of 20 μl, and Shimadzu CLASS-VP ver- Identification of Monascus purpureus MTCC sion 5.032 software, with a mobile phase made 410 of Acetonitrile/water (65:35 v/v and 3.5 pH) The morphological characteristics ofMonascus acidified with ortho-phosphoric acid (Pan- purpureus were identified by the pattern of its da et al., 2010). Citrinin concentration was then growing on potato dextrose agar. The mycelium estimated using the formula: Area of the sam- was white in the early stages; however, it rapidly ple/Area of the std × Amount of std/Dilution changed to rich pink and subsequently to dis- of std × Dilution of sample/amount of sample. tinctive yellow-orange colour (Fig. 1a, b). Deep crimson colour was formed as the culture aged Estimation of lovastatin (Patcharee et al., 2007). Stock solution of lovastatin (6 mg/ml) was prepared in ethanol using pure and standard Effect of amino acids on biomass production lovastatin gifted by Biocon, Bangalore, India. Among all 24 different amino acids tested for Hydroxylamine hydrochloride (12.5%) and biomass production, the results showed that sodium hydroxide (12.5%) both in methanol M. purpureus produced maximum fresh bio- were prepared by refluxing 12.5 g of the solid mass of 143.66 g/l with supplementation of 1% material with 100 ml of methanol for few min- D-serine (Table 1). Amino acids promoted bio- utes, then their equal volumes were mixed and mass production.

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