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1 微生物学通报 Microbiology China Sep. 20, 2014, 41(9): by Institute of Microbiology, CAS DOI: /j.microbiol.china 生物实验室用氨基葡萄糖含量的测定值间接表示红曲菌固态发酵过程中生物量的研究范海滨 1 黄常毅 2 许赣荣 1* 张薄博 1 刘飞 ( ) ( ) 2 摘要目的为准确快速地了解紫色红曲菌固态发酵中生物量的变化, 方法采用理化方法测定菌体量和氨基葡萄糖含量, 研究了不同培养时间培养基组成培养方式下菌体量与氨基葡萄糖含量的关系, 建立生物量和氨基葡萄糖含量的换算关系式 ; 构建关联该菌固态培养物近红外光谱数据与实测氨基葡萄糖含量的 PLS 模型结果建立了可通过近红外光谱法测定氨基葡萄糖来快速预测固态发酵生物量的方法, 其中最优近红外模型的校正集内部交叉验证均方根误差 (RMSECV) 为 , 预测集相关系数 (R p ) 和均方根误差 (RMSEP) 分别为和 ; 同时利用所建的换算关系式也大大提高了生物量计算的准确性结论基于所建立的生物量和氨基葡萄糖的换算关系式, 利用近红外光谱法可以快速并且较准确地测定紫色红曲菌固态发酵过程中生物量的变化关键词固态发酵, 红曲菌, 生物量, 氨基葡萄糖, 近红外光谱 The study of indirect determination of biomass in solid-state fermentation of Monascus by the measurement of glucosamine content FAN Hai-Bin 1 HUANG Chang-Yi 2 XU Gan-Rong 1* ZHANG Bo-Bo 1 LIU-Fei 2 (1. The Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi, Jiangsu , China) (2. Key Laboratory of Advanced Process Control for Light Industry, Ministry of Education, Institute of Automation, Jiangnan University, Wuxi, Jiangsu , China) Abstract: [Objective] The objective of this paper was to provide an accurate and rapid determination method for the biomass of Monascus purpureus in solid-state fermentation (SSF). [Methods] The biomass and glucosamine content was measured by physical and chemical methods, then the relation between biomass and glucosamine content under different cultivation time, media and culture methods was investigated and the conversion relation of biomass and glucosamine 基金项目 : 863 (No. 2012AA021302) * 通讯作者 : grxu123@126.com 收稿日期 : 接受日期 : 优先数字出版日期 (

2 1910 微生物学通报 Microbiol. China 2014, Vol.41, No.9 content was built. Moreover, the PLS model for the relation between near infrared spectra and measured glucosamine content of medium was built. [Results] The rapid method for estimating biomass in SSF was built by measuring glucosamine content with NIR. The root mean square error of cross-validation of calibration set (RMSECV), the prediction correlation coefficient (R p ) and root mean square error of prediction set (RMSEP) of the best model were , and , respectively. Besides, the accuracy of biomass calculation was enhanced by the conversion correlation. [Conclusion] On the base of the conversion relation of biomass and glucosamine content, the biomass of Monascus purpureus in SSF could be determined more accurately and rapidly by use of NIR method. Keywords: Solid-state fermentation, Monascus, Biomass, Glucosamine, NIR [1] Monacolin K [2] [3] [4] dos Santos [5] f c =1.78 mg/g dry mycelium P (mg/g) X=P/1.78 (g/g dry medium) Nagel [6] dos Santos Elson-Morgan [7] ( 24 h) (NIR) [8] [9] (FT-NIR) [10] FT-NIR NIR PLS (NIR-PLS) NIR 1 材料与方法 1.1 材料和仪器实验仪器及药品 : UV Hitachi FA1004 ( g) SPX-250B-Z Thermo Antaris MX 菌种 : Monascus purpureus zh 培养基 :(1)

3 : 1911 (PDA) (2) (g/l) NaNO 3 2 KH 2 PO MgSO 4 7H 2 O 1 ph 5.5 (3) (WPL ) 100 g 30 min 8 20 g 10 g NaNO 3 2 g MgSO 4 7H 2 O 1 g KH 2 PO4 1.5 g 1 L ph (4) (WPA ) [11] 100 ml 4 g min 4 mm 4 mm 4 mm 500 ml (5) (LM g/l) NaNO 3 2 MgSO 4 7H 2 O 1 KH 2 PO ph 5.0 (6) (250 ml ) 10 g 5 ml (g/l) NaNO 3 2 MgSO 4 1 KH 2 PO ph 实验方法种子液制备和发酵培养 : PDA 20 ml (10 7 /ml) 100 ml 500 ml 32 C 170 r/min h 10% WPL LM ( 100 ml/500 ml ) 32 C 170 r/min WPA 32 C 5 ml 32 C 紫色红曲菌纯菌体的制备 : WPA 400 ml 30 min 氨基葡萄糖的提取与测定 [12] : 0.1 g 0.5 g 2 ml 60% H 2 SO 4 25 C 24 h 1 mol/l H 2 SO ml Pa 1 h 1 mol/l NaOH ph ml Elson Morgan 530 nm 3 2 ml 光谱采集 : h 12 h C Thermo Antaris MX cm 1 (3 112 ) 32 8 cm 氨基葡萄糖含量近红外定量分析模型的构建 : TQ Analyst (PLS) TQ Analyst Diagnostic Spectrum Outlier Kennard-

1912 微生物学通报 Microbiol. China 2014, Vol.41, No.

1 氨基葡萄糖标准曲线的绘制 8 6 4 2 mg 100 ml 1.2.3 OD 530 X Y 1 y=9.

2 同一时间点菌体中氨基葡萄糖质量和生物量线性关系的研究 0.1 0.2 0.3 0.4 0.

996 9 图 2 菌体量和氨基葡萄糖质量的关系 Figure 2 The relation

3 培养方式培养基组成成分的种类培养时间对单位质量菌体中氨基葡萄糖含量的影响 WPL

3 3 3 3 WPL 图 1 氨基葡萄糖标准曲线 Figure 1 The standard

4 1912 微生物学通报 Microbiol. China 2014, Vol.41, No.9 Stone (K-S) [13] [14] 2 结果与分析 2.1 氨基葡萄糖标准曲线的绘制 mg 100 ml OD 530 X Y 1 y= x R 2 = 同一时间点菌体中氨基葡萄糖质量和生物量线性关系的研究 g X Y 2 2 y= x R 2 = 图 2 菌体量和氨基葡萄糖质量的关系 Figure 2 The relation of mycelial weight and glucosamine weight 2.3 培养方式培养基组成成分的种类培养时间对单位质量菌体中氨基葡萄糖含量的影响 WPL WPA LM 16 h WPL 图 1 氨基葡萄糖标准曲线 Figure 1 The standard curve of glucosamine weight 图 3 红曲菌菌体中氨基葡萄糖含量的变化 Figure 3 The changes in glucosamine content of Monascus mycelium

5 : 1913 WPA WPL LM dos Santos E. [5] dos Santos E. 2.4 发酵过程中氨基葡萄糖总量和总生物量的变化 = 2.5 麸皮固态发酵培养基培养紫色红曲菌过程中生物量的计算方法 2.4 (1) 图 4 发酵过程中生物量和氨基葡萄糖总量的变化 Figure 4 The changes in biomass and total glucosamine weight in the fermentation A WPL B WPA C LM. Note: A: WPL medium; B: WPA medium; C: LM medium. 单位质量培养物中氨基葡萄糖含量 (mg/g dry medium) 生物量 (mg/g dry medium) 单位质量菌体中氨基萄糖含量 (mg/g dry mycelium) (1)

6 1914 微生物学通报 Microbiol. China 2014, Vol.41, No.9 [15] 2.21 g/l (MPA ) MPA (1) MPA (G X ) 5 (2) 70.3 GX T 16 h R 2 =0.98 (2) 1 e T 16 h 16 h (3) 2.6 近红外光谱法测定固态培养物中氨基葡萄糖的变化 TQ Analyst Diagnostic Spectrum Outlier 9 Kennard-Stone (K-S) 90 图 5 红曲菌菌体中氨基葡萄糖含量与培养时间的拟合曲线 Figure 5 The fitted curve of glucosamine content of Monascus as a function of cultivation time cm cm 1 (MSC) Savitzky-Golay (S-G) Savitzky-Golay (SG) 单位质量培养物中氨基葡萄糖含量 (mg/g dry medium) 生物量 (mg/g dry medium) T 16 h e T (3) Process parameters Glucosamine Table 1 Sample sets 表 1 校正集与预测集氨基葡萄糖的测量结果 Actual values of glucosamine in the calibration and prediction sets No. of samples Range (mg/g dry medium) Mean (mg/g dry medium) Calibration sets Prediction sets S.D.

: 1915 图 6 样品的近红外光谱图 Figure 6 The NIR spectra of samples 14 (RMSECV) 0.

747 96 0.400 23 10.92% 0.57% 2.264% 3 讨论 49% 58% 100% 0.970 0.981 0.

NIR predicted values of glucosamine content 图 8 麸皮固态发酵培养基培养红曲菌过程中生物量的变化

7 : 1915 图 6 样品的近红外光谱图 Figure 6 The NIR spectra of samples 14 (RMSECV) (R p )(RMSEP) 独立样本预测 (3) % 0.57% 2.264% 3 讨论 49% 58% 100% [16-18] 图 7 氨基葡萄糖含量实测值与 NIR 预测值的对比 Figure 7 Actual values versus NIR predicted values of glucosamine content 图 8 麸皮固态发酵培养基培养红曲菌过程中生物量的变化 Figure 8 The variance of biomass in SSF of Monascus in wheat bran solid-state medium

8 1916 微生物学通报 Microbiol. China 2014, Vol.41, No.9 Li [19] T. reesei 3# Li T. reesei 3# 4 结论 5 min 参考文献 [1] Pandey A, Soccol CR, Mitchell D. New developments in solid state fermentation: I-bioprocesses and products[j]. Process Biochemistry, 2000, 35(10): [2],,. 3 [J]., 2006, 25(1): [3] Scotti CT, Vergoignan C, Feron G, et al. Glucosamine measurement as indirect method for biomass estimation of Cunninghamella elegans grown in solid state cultivation condition[j]. Biochemical Engineering Journal, 2001, 7(1): 1-5. [4],,,. [J]., 2005, 24(3): [5] dos Santos E, Piovan T, Roberto IC, et al. Kinetics of the solid state fermentation of sugarcane bagasse by Thermoascus aurantiacus for the production of xylanase[j]. Biotechnology Letters, 2003, 25(1): [6] Nagel FJJI, Tramper J, Bakker MSN, et al. Model for on-line moisture-content control during solid-state fermentation [J]. Biotechnology and Bioengineering, 2001, 72(2): [7],,. [J]., 2003(6): [8] Balabin RM, Safieva RZ, Lomakina EI. Comparison of linear and nonlinear calibration models based on near infrared (NIR) spectroscopy data for gasoline properties prediction[j]. Chemometrics and Intelligent Laboratory Systems, 2007, 88(2): [9],,,., [J]., 2004, 21(5): [10]. FT-NIR, [J]., 2003, 20(2): [11] Zhang BB, Lu LP, Xia Y, et al. Use of agar as carrier in solid-state fermentation for Monacolin K production by Monascus: a novel method for direct determination of biomass and accurate comparison with submerged fermentation[j]. Biochemical Engineering Journal, 2013, 80(15): [12] Sakurai Y, Lee TH, Shiota H. On the convenient method for glucosamine estimation in koji[j]. Agricultural and Biological Chemistry, 1977, 41(4): [13]. [M]. :, 2011: 4. [14],,. [J]., 2004, 37(5): [15]. [M]. :, 2005: 9. [16] Tiwari S, Suraishkumar GK, Chandavarkar A. Robust near-infra-red spectroscopic probe for dynamic monitoring of critical nutrient ratio in microbial fermentation processes[j]. Biochemical Engineering Journal, 2013, 71: [17] Blanco M, Peinado AC, Mas J. Analytical monitoring of alcoholic fermentation using NIR spectroscopy[j]. Biotechnology and Bioengineering, 2004, 88(4): [18] Liang J, Zhang D, Guo X, et al. At-line near-infrared spectroscopy for monitoring concentrations in temperature-triggered glutamate fermentation[j]. Bioprocess and Biosystems Engineering, 2013: 1-9. [19] Li HQ, Chen HZ. Near-infrared spectroscopy with a fiber-optic probe for state variables determination in solid-state fermentation[j]. Process Biochemistry, 2008, 43(5):

Microsoft Word - 793-797 tb20150504赵宏宇s-高校教改纵横.doc

Microsoft Word - 793-797 tb20150504赵宏宇s-高校教改纵横.doc 微生物学通报 Microbiology China tongbao@im.ac.cn Apr. 20, 2016, 43(4): 793 797 http://journals.im.ac.cn/wswxtbcn DOI: 10.13344/j.microbiol.china.150504 高校教改纵横生物工程专业发酵课程群建设探索 * 赵宏宇