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Proceeding of the 4 rd International Symposium“NEW RESEARCH IN BIOTECHNOLOGY” USAMV Bucharest, Romania, 2011culture media in comparison with bioproducts obtained with the same strain , but insubmerged culture (figure 4). The aim of our study is to quantify the content of azaphylonein Monascus metabolite obtained in submerged media and the citrinin, in order to indicatethe suitability to use Monascus metabolites as food additiveFig.4. Content of gamma aminobutyric acid in bioproducts obtained with Monascus sp. onsolide state or submerged biosynthesis842. MATERIAL AND METHODSIn this study we used 2 strains of Monascus: Monascus sp.1 and Monascus sp.2 supplied byBioengineering and Biotechnology Department of University POLIEHNICA of Bucharest.Metabolites from the two strains were obtained in submerged biosynthesis, with threeculture media which contain starch (Medium 1), ethanol (medium 2) and dextrose (medium3). The content of each culture media used in this study is presented in Table 1. Theazaphylone content was monitorised during all bioprocess (10 days) by measuring theoptical density of aqueous extract at 400 nm (for yellow azapylones), 420 nm (for orangeazaphylones) and 500 m respectively (for red azaphylones). All experiments was realised intriplicate, on the thermostated Heydolph rotary shaker, in Erlenmeyer flasks with totalvolume of 500 ml. Each Erlenmeyer flask was filled with 100 ml culture media. In additionfor all samples resulted from biosynthesis on dextrose media, the citrinin content wasmeasured by higy performance liquid chromaotography (HPLC). The device used was theAgilent type, with C18 column. The solvent used as carrier was the methanol. Calibrationcurve was recorded with methanolic citrinin solution (the citrinin was supplied by Sigma).3. RESULTS AND DISCUSSIONIn culture media which contain starch, Monascus sp.1 do not producedpigmentation. For the second strain Monascus sp.2, maximum concentration of red
Proceeding of the 4 rd International Symposium“NEW RESEARCH IN BIOTECHNOLOGY” USAMV Bucharest, Romania, 2011azapylones was obtained after 106 h, (figure 5) with a OD=3.8 units units, whilst maximumconcentration of orange compounds was obtained after 105 h, when a OD = 4.2 wasobtained (figure 6.)Table 1. Culture media used o obtain Monascus metabolite in submerged mediaCulture media with starchCulture media with ethanolStarch, g/L 30 Glutamic acid, g/L 5Peptone, g/L 4 K 2 HPO 4, g/L 5glutamic acid, g/L 1 KH 2 PO 4, g/L 5bidistiled water up to1L MgSO 4 •7H 2 O, g/L 0.5Culture media with dextrose CaCl 2, g/L 0.5Dextrose, g/L 30 FeSO 4 •7H 2 O), g/L 0.5NaNO 3, g/L 0.1 ZnSO 4 •7H 2 O, g/L 0.01MgSO 4, g/L 0.05 MnSO 4 •H 2 O, g/L 0.03FeSO 4 , g/L 0.01 Ethanol, g/L 20ZnSO 4 , g/L 0.08 bidistiled water up to1LMnSO 4 , g/L 0.03bidistiled waterup to1LMaximum concentration for yellow azaphylones was attain in 114 h with a OD =2.85 (figure 8.)In media with etanol the behaviour of Monascus sp 1 is the same like in the caseof starch media (without pigment production). In the case of strain Monascus sp.2 ,azaphylone appear, but in the concentration less than that obtained in starch culture media.So, in this case the productions of azaphylones red and yelow attain the maximumconcentration after 145h, corresponding to an OD=1(figure 8 and figure 10), whereas thecontent of orange azaphylone attain a maximum concentration after 135 h with an OD =0.27 (figure 9.In submerged media which contain dextrose, the both strains produce azaphylones,but content of their is less than azaphylone concentration obtained in starch or etanolculture media. The Monascus sp. 1 strain give a maximum concentration of red azaphilonesafter 160 h, when obtain an OD =0.75 (figure 11). After 164 h, a maximum concentrationof orange pigment is obtained (figure 12) with an OD =1. Two hour later (at 166 h) amaximum concentration of yellow pigment was obtained; with an OD =1 (figure 13),Monascus sp. 2 give a maximum concentration of red azaphylones after 153 h,with an OD =0.75 (figure 14). After 166 h of biosynthesis, a maximum concentration oforange pigment was obtained (figure 15) with an OD=1. After that, at 177h a maximumconcentration of yellow pigment was obtained, with an OD =1.2 (figure 16)Fungal mycotoxin formation in submerged media. Fungi from Monascus genusproduce one mycotoxin: citrinin. Content of this toxic compound in food, raw materials forfood industry or fodder depend of Monascus strains and culture media.85
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