Studies On Photostability Of Monascus Pigments

The studies on the separation and identification of monascus pigment, the mechanism of photobleaching of red, orange and yellow pigments of monascus pigments, the breeding of monascus to improve the yield and photo-stability of monascus pigments, the production of monascus pigments with photo-stability —W-red pigments by the biological modification method (i.e. separation of bean powder hydrolysate), the mechanism to increase the photo-stability of W-red pigments based on the identification of molecular structure of W-red pigments followed by irradiation were mainly carried out in this thesis. In addition, the physical and chemical photo-protection of monascus pigments was studied. The above-mentioned researches were explained as follows.Pure red pigments were separated from monascus pigment powder by extracting method with hexane, ethyl acetate and methanol in sequence. The major orange and yellow pigments were obtained by the same separation process. Further purification of orange and yellow pigments was done by HPLC. The UV-spectra of monascus pigment powder, red, orange and yellow pigments were 510 nm, 410 nm;491.5 nm, 460.5 nm;491.5 nm, 372.0 nm;and 369.0 nm, 281.5 nm, respectively.Two new red pigments, L-red pigment and W-red pigment, were determined by MS, H¹NMR and IR. For L-red pigment, its molecular formula is C₄₁H₅₅NO₁₃ with molecular weight (MW) being 768.4, and UV-spectrum is 490 nm. For W-red pigment, its molecular formula is C₂₀H₂₆N₂O₄ (MS is 364.2), and UV-spectrum is 484 nm.The photobleaching mechanism of three kinds of monascus pigments was deduced by analyzing the molecular structures of yellow and red pigments after fading. When monascus pigments are radiated with UV-light, their molecular structures are firstly decomposed into two radicals between fatty chain and benzene-cycle according to Norrish's I theory. Then, there occurred the electrons of benzene-cycle redistribution as well as the formation of new molecular. Meanwhile, a lot of radicals like OH^-, e^-_aq, etc. attacked the pigment molecular, and hence the chemical reactions happened, resulting in monascus pigment fading because covalent bond disappeared.The photo-stability of three kinds of monascus pigments was determined. Yellow pigments showed best photo-stability, the next is red pigment, and the last orange pigment. The monascus pigments should be separated firstly prior to determining its photo-stability. The testing condition is recommended as UV-lightirradiation, absorption 1.0, distance 15mm, thicker of liquid film 4.5mm, irradiation time lh. The photo-stability of L-red pigment and W-red pigment is35.7 % and 70.3%, respectively.A new Monascus strain named Monascus ruber 102, which has a higher yield of monascus pigments, was bred from Monascus ruber N by the input of N+. The promising dose is found to be 1.32-2.01 X 105 ions/s under 10 KeV. The yield of monascus pigments with Monascus ruber 102 in fermentation culture is 70.6 U/mL, 34.3% higher than that with the original strain Monascus ruber N (52.5 U/mL in this case).A new Monascus strain named Monascus ruber 102w, which could grow on substance-3 (in bean powder hydrolysate) of more than 0.1%, was screened by UV radiation. Its character of colonies was distinctively different from the original Monascus ruber 102. The production of red monascus pigment rose from 0.124 g/50mL to 0.523 g/50mL if the concentration of substance-3 increased from 0.01% to 1.6% in culture, 2.7 fold higher.The effects of standard amine acids on the color value and photo-stability of monascus pigments were studied. The results show that the photo-stability of red pigments belonging to Val, Arg and Lys groups (91.9%, 89.0% and 88.5%, respectively) is nearly 10 % higher than that of the control group (75%). However, the synthesis of monascus pigments was greatly restrained in Val group. Red monascus pigments whose MW are 364.32 and 390.31 show more photo-protective than those whose MW is 380.27. Two kinds of orange pigments, Rubropunctatin and Monascorubrine, were produced. Rubropunctatin tended to transform into Monascorubrine (red pigments). Acidity amine acids promoted more red monascus pigments than alkaline amine acids. The color values of Glu and Asp group are 82.1 U/mL and 85.6 U/mL, 97.3% and 105.7% higher than that of the control group (41.6 U/mL), respectively. The color values of Lys and Arg groups are only58.8 U/mL and 63.9 U/mL.The effect of bean powder hydrolystate on the photo-stability of red monascus pigments was studied. The results show that a new photo-protective red pigment, W-red pigment was produced in the culture of substance-3 and rice powder. Its photo-stability is 70.3 % compared to 58.3% in the control group.The photo-protection and mechanism of photo-bleaching of W-red pigment were deduced by analyzing its molecular structure after fading based on MW, H*NMR, and IR. The reason for the photo-protection of W-red pigment is that W-red pigment is short of fatty chain and combining hydroxyl thus it cannot formfree radicals.The MW of the red monascus pigment changes greatly from 905.8 without light shining to 413.6 and 803.5 with light shining during culturing. Correspondingly, the photo-stability of red moanscus pigments also increased from 64.3% to 76.4%. However, the synthesis of yellow monascus pigments was inhibited by irradiation.New red pigments formed when orange pigment reacted with Lys and Glu. Their photo-stability is 67.7% and 43.0%, and the UV-spectra are 494.5 nm, 309.5 nm and 490 nm, 305.5 nm. The ratio of orange pigment to Glu and Lys is 1:3 and 1:2, without heating in the course of reaction.The photo-stability improved by adding antioxidants such as quercetin (radical absorption), Vc (O2 elimination reagent), P-carotene (singlet O2 quenching agent) and EDTA (mental ions pincers agent). Photo-bleaching of red monascus pigments is inhibited when 0.1% Vc was added in red pigments resolvent, and the photo-stability increased from 35.7% to 87.5%.The carrier of red monascus pigments on sale is resolvable-protein, which increased the photo-stabilty of red monascus pigments from 35.0% to 54.6%. But other carriers such as sodium alginate, gelatin and |3-cycle detrin, increased the photo-stability from 35.0% to 66.5%, 66.3% and 60.7%, respectively.