Microsoft Word - cet-01.docx CHEMICAL ENGINEERING TRANSACTIONS VOL. 46, 2015 A publication of The Italian Association of Chemical Engineering Online at www.aidic.it/cet Guest Editors: Peiyu Ren, Yancang Li, Huiping Song Copyright © 2015, AIDIC Servizi S.r.l., ISBN 978-88-95608-37-2; ISSN 2283-9216 Effects of Culture Conditions on Production of Red Pigment and Citrinin by Fermentation of Monascus Ruber Zhiguo Zhou*, Hongzhen Guo, Chunyan Xie Department of Life Science and Technology, Langfang Normal University, Langfang, Hebei, 065000, China. Zhiguo_zhou@163.com Effects of carbon source, nitrogen source, and pH value and culture volume on red pigment and citrinin production by fermentation of Monascus ruber were investigated in this study. In order to improve the yield of red pigment and to reduce the production of citrinin, the four factors and three levels orthogonal experiment was used in this experiment. Results indicated that carbon source, nitrogen sou rce, pH value and culture volume influenced the production of pigment and citrinin significantly, and the red pigment with highest value was obtained at 30 g/L corn flour, 2 g/L NH4Cl, pH 4.0 and 100 ml/250ml culture volume, which also without citrinin. The results of the experiments will be helpful to the production of safe pigment, and will improve the usage of pigment in food industry. 1. Introduction Food color is an important factor related to food sensory quality. However, it usually changes during processing or in storage. The natural food pigments are safety and rich color. The microbial production of natural pigment has a great potential, and Monascus ruber has become the main direction in this area, because it is better than the natural pigments from animal and plant sources comparing the price and the stability of the pigment move (Beatriz Kilikian, et al. 2007). The French scholars Blanc and his colleagues (L. Pastrana et al. 1995) confirmed that some Monascus ruber strains can secrete toxic citrinin. The toxicity of citrinin is similar to aflatoxin B and its target is kidney organs. This finding has raised a big question on the safety of natural red pigment and red yeast rice products. Many scholars try to use different methods to control or reduce the Monascus ruber citrinin(Yu Huiling, Nie Xiaohua, 2005). However, due to the stability of the red pigment, red pigment color value has been reduced after some processing. Therefore, seeking the kind of high color and low citrinin food colorant becomes the challenge in food industry. In this paper, a liquid fermentation method is used, through inhibit the generation of citrinin and to improve the color value of red yeast rice. The study concluded that the medium composition and fermentation condition has impact on red yeast Monascus ruber generating pigment and citrinin. It determines the medium composition and fermentation conditions of the high-yielding red pigment without producing the citrinin, it provides a theoretical basis for future food security production of red pigment. 2. Materials and Methods 2.1 Strain Monascus ruber, Separated by the red heart Daqu used by the ShanXi Vinegar. 2.2 Test Equipments The ZF-III Temperature controlled bio-incubator (by XingTa Agricultural Equipment Factory); TH2 -82A model Steam and bath constant temperature oscillator (by Changzhou Guohua Electric Appliance Co., Ltd.); TD4 desktop centrifuge (by Hunan Instrument Plant centrifuge plant); the 22pc visible spectrophotometer (by Beijing Optical Instrument Factory); CS101,-2E Electric Blast Oven (by Chongqing Instrument Co., Ltd.); LC- 20 High performance liquid chromatograph (by Shimadzu, Japan) DOI: 10.3303/CET1546228 Please cite this article as: Zhou Z.G., Guo H.Z., Xie C.Y., 2015, Effects of culture conditions on production of red pigment and citrinin by fermentation of monascus ruber, Chemical Engineering Transactions, 46, 1363-1368 DOI:10.3303/CET1546228 1363 javascript:showjdsw('showjd_0','j_0') 2.3 Medium Slant preservation medium: malt extract agar (g/L): maltose 80, agar 15. The seed medium (g/L): Maltose 30 g, NaNO3 2 g, KCL 0.5 g, K2HPO4 1 g, MgSO4 0.5 g, FeSO4 0.01 g, agar 15 -20 g, distilled water 1000 ml, pH 5.8, 250 mL triangle bottled 100 mL, 21℃ sterilization 20 min, and were inoculated with activation of the good bacteria, and placing a shaking 30℃, 200r/min shaking culture for 3 days. 2.4 Monascus Ruber Culture Method (Silvana T. et al. 2008) 2.4.1 Spore Suspension of Production Sterile water pipette transferred to malt extract agar slant culture slant spores with ring vaccination stripped, dissolved in sterile water, broken up with glass beads so that the spore concentration cells of 5.0 ×10 6-5.0 ×107 / mL. 2.4.2 Monascus Ruber Seed Culture Flask of 50 mL/250 mL, and inoculated with spore suspension of 5mL, the 200r/min gas bath shaking at 30℃ for 48h. 2.4.3 Shake Flask Fermentation 2.4.3.1 Single factor carbon source fermentation Fermentation medium (g/L): carbon source, glucose, corn flour, millet flour, maltose, soluble starch as carbon source, pH 7.0, the 3% inoculum size, flask 150 mL, and the rest of the culture conditions with the seed culture medium, shaking culture for 7 days. 2.4.3.2 Fermentation on Each pH Level Medium composition with the seed culture medium, pH values were 3.5, 4.0, 5.4, 6.0, 7.0 and the remaining culture conditions with a carbon source, single-factor experiment. 2.4.3.3 Fermentation on Each Nitrogen Source Level Nitrogen sources were used to select sodium nitrate, ammonium chloride, soybean meal, peptone, glutamic acid, and the remaining ingredients with the seed culture medium, the rest of the culture conditions with the carbon source of single-factor test. 2.4.3.4 Fermentation culture on Installed Fluid Volume Level Medium composition with the seed culture medium, 250 mL flask fluid volume were 75 mL, 100 mL, 125 mL, 150 mL, 175 mL, and the remaining culture conditions with the carbon source of single-factor test, 2.4.3.5 Orthogonal Fermentation From the experiments of above single factor fermentations, choose top three levels to produce the Monascus ruber pigment. Then proceed the four factors and three levels of orthogonal experiment design. The remaining culture conditions are the same as the carbon source, single-factor test. 2.5 Determination of the Indicators and Methods 2.5.1 Color Value (QUAN Guijing et al. 2014) Water-soluble red pigment: After fermentation, the fermentation broth was filtered with four layers of gauze. Then it was diluted with the amount of distilled water and shaken. Using the distilled water as blank control, measure the OD value of 510 nm and 410 nm with a spectrophotometer. Alcohol-soluble red pigment: Place the filtered mycelium in 50℃ blast oven and bake for 15-20 min, fully ground. Use ethanol, 75% (volume fraction) of the fermentation broth, shock extraction for 24 h. Centrifugation, diluted with the amount of 75% (volume fraction) ethanol. Using the 75% (volume fraction) ethanol as the blank control, measure the OD value of 510 nm and 410 nm with a spectrophotometer. Color value = OD × dilution factor 2.5.2 Biomass (Wang Hailei, et al. 2011) Mycelium remained on the upper layers of gauze after the fermentation broth was filtered with four layers of gauze. It was washed with distilled water till the washing liquid colorless. Then the mycelia was transferred to the filter paper dried using the filter paper, weighted which is the wet bacteria weight. Biomass = weight of wet cells / fermentation liquid volume 2.5.3 Determination on content of citrinin (JyhJye Wang et al., 2004) After fermentation, mash the fermentation broth and mycelium, imbibe the 10ml of processed fermentation broth, add 20 mL of ethanol, and place them in the oscillation of the water bath for 1h, then 6000r/min centrifuge 10min, and filter through a 0.45 μm membrane for further usage. Filtrate filter with high performance liquid chromatographic is used to measure the citrinin content. HPLC chromatographic conditions are as follows column temperature: 28℃, mobile phase: (deionized water, phosphoric acid, pH3.0): V (methanol) = 47:53; fluorescence detection: λ ex = 331 nm, λ ex = 500 nm; column: 5μm; Edopse XDB Reversed - phase C. column length and column diameter: 250 mm × 4.6 mm; volume flow rate: 1.0 mL / min; sample volume: the standard sample and the sample are 20μL. 1364 3. Results and Analysis 3.1 Effects of Carbon source on the production of red pigment and citrinin Choose different carbon sources, the results are shown in Table 1. Table 1: Effects of carbon source on the production of red pigment and citrinin Carbon source Biomass g/ml Sensory color Water-soluble U/ml Alcohol-soluble U/ml Color value U/ml Citrinin mg/L Glucose 0.474 Orange 38.88 100.8 139.68 0.93 Corn flour 2.58 Crimson 129.6 157.68 573.88 0.18 Millet flour 1.46 Rose Red 100.8 136.8 237.6 - Maltose 1.56 Crimson 108.72 142.56 251.28 1.23 Soluble starch 0.23 Reddish 59.76 64.08 123.84 - Note: - expressed was not detected. The results showed that the color value of the red yeast rice is the highest if using corn meal as the sole carbon source, followed by maltose and rice flour as a carbon source. Using glucose and soluble starch as carbon source get the lowest of Monascus ruber color. The effect of Carbon sources on the production of citrinin trend is different from the one on red pigment. Using maltose as carbon source, the highest yield of citrinin is 1.23 mg / L, followed by glucose as carbon source. While using the millet flour and soluble starch as the carbon sources, no citrinin is detected. 3.2 Effects of nitrogen source on the production of red pigment and citrinin Select sodium nitrate, the results are shown in Table 2. Table 2: Effects of nitrogen sources on production of red pigment and citrinin Nitrogen source Biomass g/ml Sensory color Water-soluble U/ml Alcohol-soluble U/ml Color value U/ml Citrinin mg/L NaNO3 1.02 Reddish 24.48 61.2 85.68 - NH4Cl 0.58 Red 72.72 48.96 121.68 - soybean 0.22 Orange 38.16 65.52 103.68 1.05 Peptone 1.32 Yellow 18 34.56 52.56 1.37 Glutamate 0.08 Light yellow 5.76 10.08 15.84 - Note: - expressed was not detected. The results showed when ammonium chloride as the nitrogen sources, the amount of mycelia grows the most, and color value of the produced red pigment is the highest. When Soybean as the nitrogen sources, the mycelium grows more than others, and the color value of the produced red pigment is higher than others too. Different type of nitrogen sources can produce quite different red pigment colors. Using soybeans, Peptone and other organic nitrogen as nitrogen source, the fermentation broth is yellow. Using sodium nitrate, ammonium chloride, urea and inorganic nitrogen as the nitrogen source, the fermentation liquid became red. This is very useful for producing different colors of food coloring. 1365 The results showed Monascus ruber fails to generate the citrinin if using sodium nitrate, ammonium chloride or glutamate as nitrogen source. It is probably due to the high pH fermentation environment caused by the consumption of the ammonium salt which can significantly raise the pH value and inhibit the metabolism of citrinin formation. Using Soybean powder and peptone as the nitrogen source, citrinin is produced relative high, as 1.05 mg/L and 1.37 mg/L respectively. 3.3 Effects of pH Values on the production of red pigment and citrinin Choose different pH values, the results are shown in Table 3. Table 3: Effects of pH values on production of red pigment and citrinin pH Biomass g/ml Sensory color Water-soluble U/ml Alcohol-soluble U/ml Color value U/ml Citrinin mg/L 3.5 0.13 Light yellow 7.2 17.28 24.48 0.61 4.0 0.2 8 Orange 10.08 16.56 26.64 0.15 5.4 0.68 Reddish 30.24 36 66.24 - 6.0 0.73 Reddish 33.84 37.44 71.28 0.11 7.0 0.50 Light red 25.92 30.96 56.88 - Note: - expressed was not detected. As shown in Table 3, the most suitable pH value for forming the Monascus rubber is 6.0. The fermentation broth color is red and the color value of the red yeast rice is the highest. If pH value between 5 -7, the color value of the red yeast rice is relatively high. If pH value between 3-4, Monascus ruber color value is the lowest and the color is light. The reason may be that the pigment was stable and not easily broken down. As for whether fermentation the pH will affect the production of citrinin, the mechanism needs to be further studied. 3.4 Effects of liquid volume on the Production of red pigment and citrinin In 250 mL flask, use different of the liquid. The results are shown in Table 4. Table 4: Effects of culture volume on the production red pigment and citrinin Liquid Volume Biomass g/ml Sensory color Water-soluble U/ml Alcohol-soluble U/ml Color value U/ml Citrinin mg/L 75 0.52 Light yellow 30.24 44.08 74.32 - 100 0.61 Reddish 50.4 54 104.4 - 125 0.41 Light red 40.10 35.05 75.15 - 150 0.21 Reddish 11.12 24.30 35.42 0.12 175 0.08 Pink 18.25 12.25 30.50 - Note: - expressed not detected. Monascus ruber has the highest color value when the liquid volume is 100 mL/ 250 mL. It has highest color value for liquid volume 100 mL/ 250 mL, followed by 125 mL/ 250 mL. Since Monascus ruber is aerobic microorganisms, liquid fermentation of red yeast is aerobic fermentation with dissolved oxygen. So liquid volume has a significant impact on producing Monascus ruber pigment. With the increasing of dissolved oxygen, the production of Monascus ruber pigment increase higher and higher. There must be sufficient oxygen supply in order to improve the content of the red pigment. Within the scope of this test, the citrinin content is very low, only a small amount is detected in 150 mL/ 250 mL. 1366 3.5 The Best Ratio Test of Monascus ruber culture conditions The factor levels are shown in Table 5 and the results of the orthogonal and intuitive analysis are shown in Table 6. Table 5: Factors and levels Level Nitrogen source Carbon source pH Culture volume( ml/250ml) A B C D 1 NaNO3 Millet flour 4.0 70 2 NH4Cl Maltose 5.4 100 3 Soybean Corn starch 6 125 Table 6: Results of orthogonal and intuitive analysis No. A B C D Sensory color Biomass (g/mL) color value(U/ml) Citrinin(mg/L) 1 1 1 1 1 Orange 0.04 291.6 0.15 2 1 2 2 2 Pink 2.5 370.0 0.38 3 1 3 3 3 Light red 0.07 298.8 - 4 2 1 2 3 Crimson 2.7 179.2 - 5 2 2 3 1 Purple 1.3 587.8 - 6 2 3 1 2 Crimson 2.9 780.1 0.19 7 3 1 3 2 Orange 0.2 113.4 - 8 3 2 1 3 Purple 0.65 558 0.05 9 3 3 2 1 Pink 1.4 487.8 0. 42 K1 1057.5 484.2 1629.9 1027.8 K2 1107.9 1273.5 1034.1 1360.8 K3 1159.2 1566.9 547.2 936 k1 352.5 161.4 543.3 342.6 k2 369.3 424.5 344.7 453.6 k3 386.4 522.3 182.4 312 R 33.9 360.9 360.9 141.6 Note: - expressed not detected. According to R, carbon sources and pH have highest R values, nitrogen source has lowest R value. This indicates that the carbon source and pH have greatest impact on the red pigment, followed by liquid volume, and nitrogen source has little influence. According to the test results, the best Monascus ruber culture condition is A2B3C1D2, the 6th in Table 6, the amount of liquid NH4CL 2 g/L, corn flour 3 0 g/L, pH 4.0, installed liquid volume 100 mL/ 250mL. The results in Table 6 show whether the Monascus ruber produces citrinin depends on the culture conditions. In experimental conditions, the content of citrinin was measured as lower 1367 than 0.5 mg/L. It is in line with the relevant provisions of the content of citrinin in the Monascus ruber pigment products. 4. Discussions 4.1 Factors affecting the Generation of citrinin Professor P.J, Blanc states, whether and how much citrinin is produced depends on the amount of Monascus culture methods, culture conditions, strain and media related. The results indicate that the citrinin is not detected in the majority of culture conditions and the detected maximum value of th e citrinin is 1.37 mg/L. Patcharee Pattanagul et al. (2008) reported that Monascus ruber in potato glucose medium did not produce citrinin, but it produced higher amount of citrinin in the yeast extract medium. Zhou Bo (2009) reported that the ammonium salt has big impact on the metabolism of Monascus ruber red pigment and citrinin. 4.2 The Biological Pathways of producing citrinin Because the citrinin is the secondary metabolites in the late of Monascus fermentation production (Li Yun Yan, et al. 2000), many scientists began to study the metabolic pathways of Monascus citrinin in recent years. First of all, an acetyl coenzyme A molecule and four malonyl coenzyme A molecules condense pentanone, and then by methylation, condensation, reduction, alkylglycosylation, reduction, oxidation, dehydration and other steps, ultimately producing citrinin. Hassan Hajja (2000) reported on the analysis of red pigment and citrinin production by Monascus ruber as a function of organic acid accumulation. At present, the metabolic pathways are not yet entirely clear, in particular, one of the enzymes. If this is clarified, it can be regulated according to their metabolic mechanism of Monascus fermentation in order to improve the production of red pigment and to reduce the production of citrinin. Acknowledgements This work was financially supported by Hebei Province, the development and application of edible and medicinal mushroom resources (YF201411), And by project of Langfang Teachers University（LSZY201304）, and the important discipline of genetics in Hebei Province. References Chen Y.Z., 2003, Functional Monascus ruber pigment Fermentation. Food Science, 24 (7): 83-87. Hajjaj H., Blanc P., Groussac E., 2000, Kinetic analysis of red pigment and citrinin production by Monascus ruber as a function of organic acid accumulation. Enzyme and Microbial Technology, 27, 619 –625 Kilikian B., Eduardo M., Roberta C., 2007, Solid-state cultivation of Monascus ruber under different bioreactor scales. Abstracts Journal of Biotechnology, S133–187 Li Y.Y., Xue Q., 2000, Monascus and Citrinin. Food and Fermentation Industries, 3, 82-87 Pastrana L., Blanc P.J., Sa nterre A.L., Loret M.O., Goma G., 1995, Production of Red Pigments by Monascus ruber in Synthetic Media with a Strictly ControlledNitrogen Source, Process Biochemisitry, 30 (4). 333 -341 Pattanagul P., Pinthong R., Phianmongkhol A., 2008, Mevinolin, citrinin and pigments of adlay angkak fermented by Monascus sp. International Journal of Food Microbiology 126, 20–23 Silveira S.T., Daroit D.J., Brandelli A., 2008, Pigment production by Monascus purpureus in grape waste using factorial design. LW T 41, 170–174 Wang H.L., Ren Z.F., Li P., Gu Y.C., 2011, Improvement of the production of a red pigment in Penicillium sp. HSD07B synthesized during co-culture with Candida tropicalis Bioresource Technology, 102, 6082–6087 Wang J.J., Lee C.L., Pan T.M., 2004, Modified Mutation Method for Screening Low Citrinin-Producing Strains of Monascus purpureus on Rice Culture. Agric. Food Chem. 52, 6977-6982 Yu H.L., Nie X.H., 2005, Low citrinin red pigment liquid fermentation process. Chinese brewing, (9), 21-24. Zhou B., Zhu M.J., W ang J.F., 2009, Ammonium salt formation of the yellow pigment of the red yeast rice, red pigment and citrinin metabolism. Chongqing Institute of Technology, 23 (1), 46-53 1368