枯草芽胞杆菌固态发酵产聚-γ-谷氨酸的工艺优化
摘要
聚-γ-谷氨酸[poly-(γ-glutamic acid)，γ-PGA]是一种天然的水溶性的聚合氨基酸，具有生物可降解性，对人体和环境无毒害等优点。γ-PGA及其衍生物可应用于农业、食品、饲料、化工、材料、药物等领域，是一种极具开发前景的多功能新型生物材料。
本课题首先优化了本实验室分离的一株高产γ-PGA的枯草芽胞杆菌ME714的固态发酵培养基。运用单因素实验、正交实验以及神经网络和遗传算法进行培养工艺优化。结果表明，黄豆饼粉和麸皮是适合枯草芽胞杆菌ME714产γ-PGA的固态发酵基质，其质量比为1∶1，250mL摇瓶固体基质装量为10g；无机盐的种类和浓度为硫酸锌0.36g／kg，硫酸锰0.1g／kg，硫酸镁0.5g／kg，氯化钙0.2g／kg；谷氨酸钠31.8g／kg、尿素28.3g／kg、柠檬酸钠24g／kg、淀粉46g／kg；料水比为1∶1，初始pH值8.0，接种量8％，发酵时间36h，过程中不翻曲；优化条件下γ-PGA产量为75.3g／kg。
进一步进行了扩大培养的初步研究。珍珠岩和谷壳作为载体均可在扩大培养条件下获得较好的γ-PGA产量。谷壳作载体可以将产量提高8.6％；珍珠岩作载体时，产量提高了11.1％。
本课题以昆明小鼠为材料初步研究了添加γ-PGA的混合饲料对不同周龄的小鼠生长的影响。结果表明，添加5g／kgγ-PGA的混合饲料对5～6周龄小鼠的生长有明显的促进作用，增重作用显著；对3～4周龄小鼠，饲料中添加5g／kgγ-PGA对其生长有极显著的抑制作用，添加2.5g／kgγ-PGA对这个时期小鼠生长的影响也是显著的。
Poly (γ-glutamic acid)(γ-PGA) is a water soluble poly amino acids which have excellent characters such as completely biodegradable, non-toxic to human and environment.γ-PGA and its derivates have potential applications in a wide range of industry and agriculture such as food, cosmetics, medicine and manure. Thus this natural and multifunctional biopolymer is worth researching and developing to a commercial material.
The solid state fermentation medium ofγ-PGA biosynthesis by Bacillus subtilis ME714, which was a newly isolated strain by our lab, was optimized. Methods such as simple factor experiment, orthogonal design, RBF neural network and Genetic algorithm were involved in this work. The higherγ-PGA yield was achieved in the conditions that described as following (g/kg): soybean cake powder and wheat bran (5∶5 w/w) as the mixed substrates supplemented with sodium glutamate (31.8 g/kg), urea(28.3 g/kg), trisodium citrate (24 g/kg), starch 46 (g/kg) and mineral salts (ZnSO_4 0.36 g/kg, MnSO_4 0.1 g/kg, MgSO_4 0.5 g/kg, CaCl_2 0.2 g/kg), with initial ratio of substrate weight (g) and solution volume (mL) 1∶1, initial pH 8.0, inoculum level 8%, at 37℃incubated 36h. No substrate disturbance was made during the cultivation. The yield is 75.3 g/kg, which is increased by 43.16 % compared to the original medium.
Scale-up ofγ-PGA biosynthesis was devolped. Both perlite and chaff as carriers have well performance during scale-up cultivation ofγ-PGA. The yield is increased by 8.6%, when chaff was applied, 11.1% for perlite, compared to original culture.
In this research, effect ofγ-PGA concentration in the animal feed on Kunming mice growth was investigated. The results showed animal feed containing 5 g/kgγ-PGA had markedly increased the weight of 5～6 weeks old mice, while inhibited the growth of 3～4 weeks old mice. Feeding with 2.5 g/kgγ-PGA also has statistical reverse effect on the growth of 3～4 weeks old mice
本课题首先优化了本实验室分离的一株高产γ-PGA的枯草芽胞杆菌ME714的固态发酵培养基。运用单因素实验、正交实验以及神经网络和遗传算法进行培养工艺优化。结果表明，黄豆饼粉和麸皮是适合枯草芽胞杆菌ME714产γ-PGA的固态发酵基质，其质量比为1∶1，250mL摇瓶固体基质装量为10g；无机盐的种类和浓度为硫酸锌0.36g／kg，硫酸锰0.1g／kg，硫酸镁0.5g／kg，氯化钙0.2g／kg；谷氨酸钠31.8g／kg、尿素28.3g／kg、柠檬酸钠24g／kg、淀粉46g／kg；料水比为1∶1，初始pH值8.0，接种量8％，发酵时间36h，过程中不翻曲；优化条件下γ-PGA产量为75.3g／kg。
进一步进行了扩大培养的初步研究。珍珠岩和谷壳作为载体均可在扩大培养条件下获得较好的γ-PGA产量。谷壳作载体可以将产量提高8.6％；珍珠岩作载体时，产量提高了11.1％。
本课题以昆明小鼠为材料初步研究了添加γ-PGA的混合饲料对不同周龄的小鼠生长的影响。结果表明，添加5g／kgγ-PGA的混合饲料对5～6周龄小鼠的生长有明显的促进作用，增重作用显著；对3～4周龄小鼠，饲料中添加5g／kgγ-PGA对其生长有极显著的抑制作用，添加2.5g／kgγ-PGA对这个时期小鼠生长的影响也是显著的。
Poly (γ-glutamic acid)(γ-PGA) is a water soluble poly amino acids which have excellent characters such as completely biodegradable, non-toxic to human and environment.γ-PGA and its derivates have potential applications in a wide range of industry and agriculture such as food, cosmetics, medicine and manure. Thus this natural and multifunctional biopolymer is worth researching and developing to a commercial material.
The solid state fermentation medium ofγ-PGA biosynthesis by Bacillus subtilis ME714, which was a newly isolated strain by our lab, was optimized. Methods such as simple factor experiment, orthogonal design, RBF neural network and Genetic algorithm were involved in this work. The higherγ-PGA yield was achieved in the conditions that described as following (g/kg): soybean cake powder and wheat bran (5∶5 w/w) as the mixed substrates supplemented with sodium glutamate (31.8 g/kg), urea(28.3 g/kg), trisodium citrate (24 g/kg), starch 46 (g/kg) and mineral salts (ZnSO_4 0.36 g/kg, MnSO_4 0.1 g/kg, MgSO_4 0.5 g/kg, CaCl_2 0.2 g/kg), with initial ratio of substrate weight (g) and solution volume (mL) 1∶1, initial pH 8.0, inoculum level 8%, at 37℃incubated 36h. No substrate disturbance was made during the cultivation. The yield is 75.3 g/kg, which is increased by 43.16 % compared to the original medium.
Scale-up ofγ-PGA biosynthesis was devolped. Both perlite and chaff as carriers have well performance during scale-up cultivation ofγ-PGA. The yield is increased by 8.6%, when chaff was applied, 11.1% for perlite, compared to original culture.
In this research, effect ofγ-PGA concentration in the animal feed on Kunming mice growth was investigated. The results showed animal feed containing 5 g/kgγ-PGA had markedly increased the weight of 5～6 weeks old mice, while inhibited the growth of 3～4 weeks old mice. Feeding with 2.5 g/kgγ-PGA also has statistical reverse effect on the growth of 3～4 weeks old mice
引文
1．卫功元，李寅，堵国成，陈坚．Candida utilis生物合成谷胱甘肽的营养及环境条件．应用与环境生物学报，2003，9(6)：642～646
2．马雷，张蓓，武改红，张克旭，陈宁．基于BP神经网络与遗传算法的鸟苷发酵培养基优化．天津科技大学学报，2005，20(3)：17-19
3．戈进杰．生物降解高分子材料及其应用．北京：化学工业出版社，2002，26-35
4．方柏山，陈宏文，谢晓兰．基于神经网络和遗传算法的木糖醇发酵培养基优化研究．生物工程学报，2000，16(3)：648-650
5．王传海，何都良，郑有飞，姚克敏，徐红．保水剂新材料γ-聚谷氨酸的吸水性能和生物学效应的初步研究．中国农业气象，2004，25(2)：19-22
6．朱玉华．MATLAB在过程参数优化中的应用．机床与液压，2004，11：165-166
7．何小兵．生物合成聚γ-谷氨酸(钠盐型)的溶液性质研究．[硕士学位论文]．南京：南京工业大学图书馆，2003
8．张礼星．固态发酵纤维素酶的研究．[博士学位论文]．无锡：无锡轻工大学图书馆，1999．
9．张俐娜，薛奇，莫志深，金熹高．高分子物理近代研究方法．武汉：武汉大学出版社，2006
10．张慧，王建，陈宁．L-缬氨酸发酵培养基的神经网络建模与遗传算法优化．生物技术通讯，2005，16(2)：156-158
11．李绍新，邢达，秦华明，杨湘波，谭石慈．油脂降解培养基优化的遗传算法实验研究．分析化学，2004，32(4)：481-484
12．杨革，陈坚，曲音波，伦世仪．金属离子对地衣芽胞杆菌合成多聚γ—谷氨酸的影响．生物工程学报，2001，17(6)：706-709
13．杨革，陈坚，曲音波，伦世仪．碳源和Mn~(-2+)对地衣芽胞杆菌Bacillus licheniformisWBL—3生产聚γ—谷氨酸的影响．化工学报，2002，53(3)：317-320
14．沙长青，李伟群，赵晓宁，王佳龙，陈静宇，杨志兴．纳豆芽胞杆菌(Bacillus subtilis natto)固体发酵生产γ-PGA．中国生物工程杂志，2004，24(10)：70-73
15．陈宏文，方柏山，胡宗定．遗传算法应用于分批发酵动力学模型参数估算．华侨大学学报，2000．21(1)：71-75
16．陈雄．枯草芽胞杆菌高产聚-γ-谷氨酸及其应用研究．[博十学位论文]．武汉：华中农业大学图书馆，2005
17．罗建平，罗凯，陈晓燕．用神经网络和遗传算法优化怀槐悬浮细胞合成异黄酮．生物工程学报，2004，20(5)：759-763
18．姚汝华．微生物工程工艺原理．广州：华南理工大学出版社，2003
19．施庆珊，许虹，林小平，邱玉棠．γ-多聚谷氨酸的微生物合成．生物技术，2004，14：65-67
20．赵斌，何绍江．微生物学实验．北京：科学出版社，2002
21．章克昌．酒精与蒸馏酒工艺学．北京：中国轻工业出版社，1995
22．诸葛健．工业微生物育种学．北京：化学工业出版社，1998
23．彭银仙，徐虹，陈国广，韦萍，欧阳平凯，张钧寿．新型药物载体聚谷氨酸的合成及其应用．中国新药杂志，2002，11：515-519
24．游庆红，张新民，陈国广，徐虹，欧阳平凯．γ-聚谷氨酸的生物合成及应用．现代化工，2002，12：56-59
25．蔡宇杰，诸葛斌，张锡红，须文波．遗传算法与神经网络耦联法优化生淀粉酶发酵培养基．无锡轻工大学学报，2001，20(4)：421-423
26. Ashiuchi M, Nakamura H, Yamamoto T, Kamei T, Soda K, Park C, Sung M H, Yagi T, Misono H. Poly-γ-glutamate depolymerade of Bacillus subtilis: production, simple purification and substrate selectivity. J Mol Cat B: Enzym, 2003b, 23: 249-255
27. Broomhead D S, Lowe D. Multivariable functional imterpolation and adaptive networks. Complex syst , 1988, 2: 321-355
28. Chen X, Chen S W, Sun M, Yu Z N. High yield of poly-gamma-glutamic acid from Bacillus subtilis by solid-state fermentation using swine manure as the basis of a solid substrate. Bioreso ur Technol, 2005, 96(17): 1872-1879.
29. Chen X, Chen S W, Sun M, Yu Z N. Medium optimization by response surface methodology for poly-gamma-glutamic acid production using dairy manure as the basis of a solid substrate. Appl Microbiol Biotechnol, 2005, 69(4): 390-396
30. Cromwick A M, Gross R A. Effects of manganese (Ⅱ) on Bacillus licheniformis ATCC 9945A physiology and γ-poly (glutamic acid) formation. Int J Biol Macromol, 1995a, 17: 259-267
31. Cromwick A M, Gross R A. Investigation by NMR of metabolic routes to bacterial γ-poly (glutamic acid) using ~(13)C-labeled citrate and glutamate as media carbon sources. Can J Microbiol, 1995b, 41: 902-909
32. Do J H, Chang H N, Lee S Y. Efficient recovery of γ-poly (glutamic Acid) from highly viscous culture broth. Biotech Bioeng, 2001, 76: 219-223
33. Doelle H W, Mitchell D A, Rolz C E. Solid Substrate Cultivation. Elsiever Sci. P.ubl. Itd; London & New York; 1992.
34. Durand A. Bioreactor designs for solid state fermentation. Biochem Eng J, 2003, 13: 113-125
35. Freyer St, Weuster-Botz D, Wandrey C. Medium optimization using genetic algorithms. Bio Eng, 1992,8(5): 16-25
36. Gonzales D, Fan K, Sevoian M. Synthesis and swelling characterizations of a poly-(gamma-glutamic acid) hydrogen. J polym Sci Part A: polym chem, 1996,34:2019-2027
37. Gross R A, McCarthy S P, Shah D T. Gamma-poly (glutamic acid) esters. US Patent, 5378807. 1995
38. Hasebe K, Inagaki S. Preparation composition for external use containing gamma- polyglutamic acid and vegetable extract in combination. JP patent 11240827, 1999-09-20
39. Ho N C, Sang Y L, Jin H D. Process for preparing gamma-polyglutamic acid from high-viscouse culture broth. US patent0016341A1. 2001-08-23
40. Karasawa M, Tanimoto S, Kouji K. The use of poly-gamma-glutamic acid for preparing an agent for increasing the gamma-glutamic acid for preparing an agent for increasing the phosphorus assimilation. EP0838160, 1998-05-06.
41. Katsifas E A, Giannoutsou E, Lambraki M, Barla M, Karagouni A D. Chromium recycling of tannery waste through microbial fermentation. J Ind Microbiol Biotechnol, 2004, 31: 57-62
42. King E C. Watkins W J. The use of poly- γ -D-glutamic acid from Bacillus lichemiformis. J Polym. Sci. Part A: Polym. Chem, 1998, 36: 1995-1999.
43. Ko Young H, Gross Richard A. Effects of glucose and glycerol on gamma-poly (glutamic acid) formation by Bacillus licheniforms ATCC9945a. Biotechnol Bioeng, 1998, 57:430-437
44. Konno A, Taguchi T, Yamaguchi T. Bakery products and noodles containing poly (γ-glutamic acid). US patent, 4888193, 1989-12-19
45. Kunioka M, Biosynthesis of poly (γ-glutamic acid) from L-glutamic acid, citric acid, and ammonium sulfate in Bacillus subtilis IFO3335. Appl Microbiol Biotechnol, 1995, 44: 501-506
46. Kunioka M, Goto A. Productioin of poly-glutamic acid by fed-batch culture of Bacillus licheniformis. Appl Microbiol Biotechnol, 1994, 40: 867-874
47. Kunioka M. Biosynthesis and chemical reactions of poly (amino acid) from microorganisms. Appl Microbiol Biotechnol, 1997, 47: 469-475
48. Lonosane B K. Optimal tuning of PI speed controller coefficients for electric drives using neural network and genetic algorithms. Enzyme microb.Technol.1985, 7: 258-265
49. Makoto T, Kouji K, Ayako S, Xu P, Ken-Ichi F, Toshio T, Yutaka T, Eiji H. Physicochemical properties of cross-linked poly-γ-glutamic acid and its flocculating activity against kaolin suspension. J biosci bioeng 2005a, 99: 130-135
50. Makoto T, Kouji K, Osamu M, Xu P, Hirokazu N, Yoshinosuke U, Akio I, Ken-ichi F, Toshio T, Yutaka T, Eiji H. Flocculating activity of cross-linked poly-glutamic acid against bentonite and Escherichia coil suspension pretreated with FeCl~3 and its interaction with Fe~(3+). J biosci bioeng 2005b, 100:207-211
51. McLean R J C, Beauchemin D, Beveridge T J. Influence of oxidation state on iron binding by Bacillus licheniformis capsule. Appl Environ Microbiol, 1992, 58: 405-408
52. Mclean R J C, Beauchemin D, Clapham L, Beueridge T J. Metal-binding characteristics of the gamma-glutamyl capsular polymer of Bacillus licheniformis ATCC 9945. Appl Environ Microbiol, 1990,59:3671-3677
53. Min X L, Liu G H. The combination of artificial neural network and genetic algorithm applied to modeling and optimization. Appl Res Comput, 2002, 1: 79-81
54. Mitchell D A., Krieger N, Stuart D M., Pandey A. New developments in solid-state fermentationII. Rational approaches to the design, operation and scale-up of bioreactors. Proc Biochem, 2000,35: 1211-1225
55. Mitsuikzi M, Mizuno A, Tanimoto H, Motoki M. Relationship between the Antifreeze Antivites and the Chemical Structures of Oligo-ang Poly(glutamic acid)s. J Agric Food Chem, 1998, 46: 891-895
56. Otani Y, Tabata Y, Ikada Y. Effects of additives on gelation and tissue adhesion of gelatin-poly (L-glutamic acid) mixture. Biomaterials, 1998, 19: 2167-2173
57. Pandey A, Carlos R, Mitchell D. New developments in solid state fermentation: I-bioprocesses and products. Proc Biochem , 2000 ,35: 1153-1169
58. Powell M J D.Radial basis functions for multivariable interpolation. In: 1MG Conference on Algorithms for the Approximation of Function and Data. UK:shrivenham, 1985, 143-167
59. Prado F C, Vandenberghe LPS, Lisboa C, Paca J, Pandey A, Soccol C R. Relation between Citric Acid Production and Respiration Rate of Aspergillus niger in Solid-State Fermentation, Eng Life Sci, 2004, 4(2): 179-186
60. Raghavarao, K S M S, Ranganathan, T V, Karanth, N G. Some engineeringaspects of solid-state fermentation. Biochem Eng J, 2003, 13 : 127-135
61. Robinson T D, Nigam S P. Solid-state fermentation: a promising microbial technology for secondary metabolite production. Appl Microbiol Biotechnol, 2001, 55: 284-289
62. Robinson T D, Nigam S P. Bioreactor design for protein enrichment of agricultural residues by solid state fermentation. Biochem EngJ, 2003, 13: 197-203
63. Saucedo-Castaneda G, Lonsane B K, Krishnaiah M M, Navarro J M, Roussos S, Raimbault M. Maintenance of heat and water balances as a scale-up criterion for the production of ethanol by Schwanniomyces castellii in a solid state fermentation system. Proc Biochem, 1992b, 27: 97-107
64. Saucedo-Castaneda G, Lonsane B K, Navarro J M, Roussos S , Raimbault M. Potential of using a simple fermenter for biomass built up, starch hydrolysis and ethanol production: Solid state fermentation system involving Schwanniomyces castellii, Appl Biochem Biotech, 1992, 36: 47-61.
65. Selvakumar P, Ashakumary L, Pandey A. Biosynthesis of glucoamylase from Aspergillus niger by solid-state fermentation using tea waste as the basis of a solid substrate. Biores Technol, 1998, 65: 83-85
66. Seydi V U, Metin D. Optimal tuning of PI speed controller coefficients for electric drives using neural network and genetic algorithms. Elect Eng, 2005, 87-77
67. Shih I L, Van Y T, Yeh L C, Lin H G, Chang Y N. Production of a biopolymer flocculant from Bacillus licheniformis and its flocculation properties. Bioresource Technology. 2001 , 78: 267-272
68. Tanimoto H, Mori M, Motoli M, Torii K, Kadowaki M, Noguchi T. Natto mucilage containing poly-γ-glutamic acid increases soluble calcium in the rat small intestine. Biosci Biotechnol Biochem, 2001,65:516-521
69. Tanimoto H, Sato H, Karasawa M, Iwasaki K, Oshima A, Adachi S. Feed composition containing poly-γ-glutamic acid. JP Patent WO9635339, 2000
70. Tanimoto H, Sato H, Kuraishi C, Kido K, Seguto K. High absorption mineral-containing composition and foods . US patent, US5447732, 1995.
71. Tanimto H, Sato H, Karasawa M, Iwasaki K, Oshima A, Adachi S. Feed composition containing poly-γ-glutamic acid. European patent, EP0826310A1, 1998-04-03.
72. Thorne C B, Gomez C G, Noyes H E, Housewright R D. Production of glutamyl polypeptide by Bacillus subtilis. J Bacteriol, 1954, 68: 307-15
73. Troy F A. Chemistry and biosynthesis of the poly (γ-D-glutamyl) capsule in Bacillus licheniformis properties of the membrane-mediated biosynthetic reaction. J Biol Chem, 1973, 248:305-315
74. Udo Ho¨ Iker and Ju¨ rgen Lenz, Solid-state fermentation — are there any biotechnological advantages? Curr Opin Microbiol, 2005, 8: 301-306
75. Xu J, Chen S W, Yu Z N. Optimization of process parameters for poly g-glutamate production under solid state fermentation from Bacillus subtilis CCTCC202048.Proc Biochem, 2005, 40: 3075-3081
76. Yokoi H, Arima T, Hirose J, Hayashi S, Takasaki Y. Flocculation properties of polyglutamic acid produced by Bacillus subtilis. J Ferment Bioeng, 1996, 82: 84-87
77. Yokoi H, Yoshida T, Mori S, Hirose J, Hayashi S, Takasaki Y. Biopolymer flocculant produced by an Enterobacter sp. Biotechnol Lett, 1996, 19: 569-573
78. Yoon S H, Do J H, Lee S Y, Chung H N. Production of Poly-γ-Glutamic Acid by fed-batch Culture of Bacillus licheniformis. Biotechnol Lett, 2000, 22: 585-588
79. Zuo K, Wu W T. Semi-realtime optimization and control of a fed-batch fermentation system. Computers Chem Eng, 2000, 24: 1105-1109
2．马雷，张蓓，武改红，张克旭，陈宁．基于BP神经网络与遗传算法的鸟苷发酵培养基优化．天津科技大学学报，2005，20(3)：17-19
3．戈进杰．生物降解高分子材料及其应用．北京：化学工业出版社，2002，26-35
4．方柏山，陈宏文，谢晓兰．基于神经网络和遗传算法的木糖醇发酵培养基优化研究．生物工程学报，2000，16(3)：648-650
5．王传海，何都良，郑有飞，姚克敏，徐红．保水剂新材料γ-聚谷氨酸的吸水性能和生物学效应的初步研究．中国农业气象，2004，25(2)：19-22
6．朱玉华．MATLAB在过程参数优化中的应用．机床与液压，2004，11：165-166
7．何小兵．生物合成聚γ-谷氨酸(钠盐型)的溶液性质研究．[硕士学位论文]．南京：南京工业大学图书馆，2003
8．张礼星．固态发酵纤维素酶的研究．[博士学位论文]．无锡：无锡轻工大学图书馆，1999．
9．张俐娜，薛奇，莫志深，金熹高．高分子物理近代研究方法．武汉：武汉大学出版社，2006
10．张慧，王建，陈宁．L-缬氨酸发酵培养基的神经网络建模与遗传算法优化．生物技术通讯，2005，16(2)：156-158
11．李绍新，邢达，秦华明，杨湘波，谭石慈．油脂降解培养基优化的遗传算法实验研究．分析化学，2004，32(4)：481-484
12．杨革，陈坚，曲音波，伦世仪．金属离子对地衣芽胞杆菌合成多聚γ—谷氨酸的影响．生物工程学报，2001，17(6)：706-709
13．杨革，陈坚，曲音波，伦世仪．碳源和Mn~(-2+)对地衣芽胞杆菌Bacillus licheniformisWBL—3生产聚γ—谷氨酸的影响．化工学报，2002，53(3)：317-320
14．沙长青，李伟群，赵晓宁，王佳龙，陈静宇，杨志兴．纳豆芽胞杆菌(Bacillus subtilis natto)固体发酵生产γ-PGA．中国生物工程杂志，2004，24(10)：70-73
15．陈宏文，方柏山，胡宗定．遗传算法应用于分批发酵动力学模型参数估算．华侨大学学报，2000．21(1)：71-75
16．陈雄．枯草芽胞杆菌高产聚-γ-谷氨酸及其应用研究．[博十学位论文]．武汉：华中农业大学图书馆，2005
17．罗建平，罗凯，陈晓燕．用神经网络和遗传算法优化怀槐悬浮细胞合成异黄酮．生物工程学报，2004，20(5)：759-763
18．姚汝华．微生物工程工艺原理．广州：华南理工大学出版社，2003
19．施庆珊，许虹，林小平，邱玉棠．γ-多聚谷氨酸的微生物合成．生物技术，2004，14：65-67
20．赵斌，何绍江．微生物学实验．北京：科学出版社，2002
21．章克昌．酒精与蒸馏酒工艺学．北京：中国轻工业出版社，1995
22．诸葛健．工业微生物育种学．北京：化学工业出版社，1998
23．彭银仙，徐虹，陈国广，韦萍，欧阳平凯，张钧寿．新型药物载体聚谷氨酸的合成及其应用．中国新药杂志，2002，11：515-519
24．游庆红，张新民，陈国广，徐虹，欧阳平凯．γ-聚谷氨酸的生物合成及应用．现代化工，2002，12：56-59
25．蔡宇杰，诸葛斌，张锡红，须文波．遗传算法与神经网络耦联法优化生淀粉酶发酵培养基．无锡轻工大学学报，2001，20(4)：421-423
26. Ashiuchi M, Nakamura H, Yamamoto T, Kamei T, Soda K, Park C, Sung M H, Yagi T, Misono H. Poly-γ-glutamate depolymerade of Bacillus subtilis: production, simple purification and substrate selectivity. J Mol Cat B: Enzym, 2003b, 23: 249-255
27. Broomhead D S, Lowe D. Multivariable functional imterpolation and adaptive networks. Complex syst , 1988, 2: 321-355
28. Chen X, Chen S W, Sun M, Yu Z N. High yield of poly-gamma-glutamic acid from Bacillus subtilis by solid-state fermentation using swine manure as the basis of a solid substrate. Bioreso ur Technol, 2005, 96(17): 1872-1879.
29. Chen X, Chen S W, Sun M, Yu Z N. Medium optimization by response surface methodology for poly-gamma-glutamic acid production using dairy manure as the basis of a solid substrate. Appl Microbiol Biotechnol, 2005, 69(4): 390-396
30. Cromwick A M, Gross R A. Effects of manganese (Ⅱ) on Bacillus licheniformis ATCC 9945A physiology and γ-poly (glutamic acid) formation. Int J Biol Macromol, 1995a, 17: 259-267
31. Cromwick A M, Gross R A. Investigation by NMR of metabolic routes to bacterial γ-poly (glutamic acid) using ~(13)C-labeled citrate and glutamate as media carbon sources. Can J Microbiol, 1995b, 41: 902-909
32. Do J H, Chang H N, Lee S Y. Efficient recovery of γ-poly (glutamic Acid) from highly viscous culture broth. Biotech Bioeng, 2001, 76: 219-223
33. Doelle H W, Mitchell D A, Rolz C E. Solid Substrate Cultivation. Elsiever Sci. P.ubl. Itd; London & New York; 1992.
34. Durand A. Bioreactor designs for solid state fermentation. Biochem Eng J, 2003, 13: 113-125
35. Freyer St, Weuster-Botz D, Wandrey C. Medium optimization using genetic algorithms. Bio Eng, 1992,8(5): 16-25
36. Gonzales D, Fan K, Sevoian M. Synthesis and swelling characterizations of a poly-(gamma-glutamic acid) hydrogen. J polym Sci Part A: polym chem, 1996,34:2019-2027
37. Gross R A, McCarthy S P, Shah D T. Gamma-poly (glutamic acid) esters. US Patent, 5378807. 1995
38. Hasebe K, Inagaki S. Preparation composition for external use containing gamma- polyglutamic acid and vegetable extract in combination. JP patent 11240827, 1999-09-20
39. Ho N C, Sang Y L, Jin H D. Process for preparing gamma-polyglutamic acid from high-viscouse culture broth. US patent0016341A1. 2001-08-23
40. Karasawa M, Tanimoto S, Kouji K. The use of poly-gamma-glutamic acid for preparing an agent for increasing the gamma-glutamic acid for preparing an agent for increasing the phosphorus assimilation. EP0838160, 1998-05-06.
41. Katsifas E A, Giannoutsou E, Lambraki M, Barla M, Karagouni A D. Chromium recycling of tannery waste through microbial fermentation. J Ind Microbiol Biotechnol, 2004, 31: 57-62
42. King E C. Watkins W J. The use of poly- γ -D-glutamic acid from Bacillus lichemiformis. J Polym. Sci. Part A: Polym. Chem, 1998, 36: 1995-1999.
43. Ko Young H, Gross Richard A. Effects of glucose and glycerol on gamma-poly (glutamic acid) formation by Bacillus licheniforms ATCC9945a. Biotechnol Bioeng, 1998, 57:430-437
44. Konno A, Taguchi T, Yamaguchi T. Bakery products and noodles containing poly (γ-glutamic acid). US patent, 4888193, 1989-12-19
45. Kunioka M, Biosynthesis of poly (γ-glutamic acid) from L-glutamic acid, citric acid, and ammonium sulfate in Bacillus subtilis IFO3335. Appl Microbiol Biotechnol, 1995, 44: 501-506
46. Kunioka M, Goto A. Productioin of poly-glutamic acid by fed-batch culture of Bacillus licheniformis. Appl Microbiol Biotechnol, 1994, 40: 867-874
47. Kunioka M. Biosynthesis and chemical reactions of poly (amino acid) from microorganisms. Appl Microbiol Biotechnol, 1997, 47: 469-475
48. Lonosane B K. Optimal tuning of PI speed controller coefficients for electric drives using neural network and genetic algorithms. Enzyme microb.Technol.1985, 7: 258-265
49. Makoto T, Kouji K, Ayako S, Xu P, Ken-Ichi F, Toshio T, Yutaka T, Eiji H. Physicochemical properties of cross-linked poly-γ-glutamic acid and its flocculating activity against kaolin suspension. J biosci bioeng 2005a, 99: 130-135
50. Makoto T, Kouji K, Osamu M, Xu P, Hirokazu N, Yoshinosuke U, Akio I, Ken-ichi F, Toshio T, Yutaka T, Eiji H. Flocculating activity of cross-linked poly-glutamic acid against bentonite and Escherichia coil suspension pretreated with FeCl~3 and its interaction with Fe~(3+). J biosci bioeng 2005b, 100:207-211
51. McLean R J C, Beauchemin D, Beveridge T J. Influence of oxidation state on iron binding by Bacillus licheniformis capsule. Appl Environ Microbiol, 1992, 58: 405-408
52. Mclean R J C, Beauchemin D, Clapham L, Beueridge T J. Metal-binding characteristics of the gamma-glutamyl capsular polymer of Bacillus licheniformis ATCC 9945. Appl Environ Microbiol, 1990,59:3671-3677
53. Min X L, Liu G H. The combination of artificial neural network and genetic algorithm applied to modeling and optimization. Appl Res Comput, 2002, 1: 79-81
54. Mitchell D A., Krieger N, Stuart D M., Pandey A. New developments in solid-state fermentationII. Rational approaches to the design, operation and scale-up of bioreactors. Proc Biochem, 2000,35: 1211-1225
55. Mitsuikzi M, Mizuno A, Tanimoto H, Motoki M. Relationship between the Antifreeze Antivites and the Chemical Structures of Oligo-ang Poly(glutamic acid)s. J Agric Food Chem, 1998, 46: 891-895
56. Otani Y, Tabata Y, Ikada Y. Effects of additives on gelation and tissue adhesion of gelatin-poly (L-glutamic acid) mixture. Biomaterials, 1998, 19: 2167-2173
57. Pandey A, Carlos R, Mitchell D. New developments in solid state fermentation: I-bioprocesses and products. Proc Biochem , 2000 ,35: 1153-1169
58. Powell M J D.Radial basis functions for multivariable interpolation. In: 1MG Conference on Algorithms for the Approximation of Function and Data. UK:shrivenham, 1985, 143-167
59. Prado F C, Vandenberghe LPS, Lisboa C, Paca J, Pandey A, Soccol C R. Relation between Citric Acid Production and Respiration Rate of Aspergillus niger in Solid-State Fermentation, Eng Life Sci, 2004, 4(2): 179-186
60. Raghavarao, K S M S, Ranganathan, T V, Karanth, N G. Some engineeringaspects of solid-state fermentation. Biochem Eng J, 2003, 13 : 127-135
61. Robinson T D, Nigam S P. Solid-state fermentation: a promising microbial technology for secondary metabolite production. Appl Microbiol Biotechnol, 2001, 55: 284-289
62. Robinson T D, Nigam S P. Bioreactor design for protein enrichment of agricultural residues by solid state fermentation. Biochem EngJ, 2003, 13: 197-203
63. Saucedo-Castaneda G, Lonsane B K, Krishnaiah M M, Navarro J M, Roussos S, Raimbault M. Maintenance of heat and water balances as a scale-up criterion for the production of ethanol by Schwanniomyces castellii in a solid state fermentation system. Proc Biochem, 1992b, 27: 97-107
64. Saucedo-Castaneda G, Lonsane B K, Navarro J M, Roussos S , Raimbault M. Potential of using a simple fermenter for biomass built up, starch hydrolysis and ethanol production: Solid state fermentation system involving Schwanniomyces castellii, Appl Biochem Biotech, 1992, 36: 47-61.
65. Selvakumar P, Ashakumary L, Pandey A. Biosynthesis of glucoamylase from Aspergillus niger by solid-state fermentation using tea waste as the basis of a solid substrate. Biores Technol, 1998, 65: 83-85
66. Seydi V U, Metin D. Optimal tuning of PI speed controller coefficients for electric drives using neural network and genetic algorithms. Elect Eng, 2005, 87-77
67. Shih I L, Van Y T, Yeh L C, Lin H G, Chang Y N. Production of a biopolymer flocculant from Bacillus licheniformis and its flocculation properties. Bioresource Technology. 2001 , 78: 267-272
68. Tanimoto H, Mori M, Motoli M, Torii K, Kadowaki M, Noguchi T. Natto mucilage containing poly-γ-glutamic acid increases soluble calcium in the rat small intestine. Biosci Biotechnol Biochem, 2001,65:516-521
69. Tanimoto H, Sato H, Karasawa M, Iwasaki K, Oshima A, Adachi S. Feed composition containing poly-γ-glutamic acid. JP Patent WO9635339, 2000
70. Tanimoto H, Sato H, Kuraishi C, Kido K, Seguto K. High absorption mineral-containing composition and foods . US patent, US5447732, 1995.
71. Tanimto H, Sato H, Karasawa M, Iwasaki K, Oshima A, Adachi S. Feed composition containing poly-γ-glutamic acid. European patent, EP0826310A1, 1998-04-03.
72. Thorne C B, Gomez C G, Noyes H E, Housewright R D. Production of glutamyl polypeptide by Bacillus subtilis. J Bacteriol, 1954, 68: 307-15
73. Troy F A. Chemistry and biosynthesis of the poly (γ-D-glutamyl) capsule in Bacillus licheniformis properties of the membrane-mediated biosynthetic reaction. J Biol Chem, 1973, 248:305-315
74. Udo Ho¨ Iker and Ju¨ rgen Lenz, Solid-state fermentation — are there any biotechnological advantages? Curr Opin Microbiol, 2005, 8: 301-306
75. Xu J, Chen S W, Yu Z N. Optimization of process parameters for poly g-glutamate production under solid state fermentation from Bacillus subtilis CCTCC202048.Proc Biochem, 2005, 40: 3075-3081
76. Yokoi H, Arima T, Hirose J, Hayashi S, Takasaki Y. Flocculation properties of polyglutamic acid produced by Bacillus subtilis. J Ferment Bioeng, 1996, 82: 84-87
77. Yokoi H, Yoshida T, Mori S, Hirose J, Hayashi S, Takasaki Y. Biopolymer flocculant produced by an Enterobacter sp. Biotechnol Lett, 1996, 19: 569-573
78. Yoon S H, Do J H, Lee S Y, Chung H N. Production of Poly-γ-Glutamic Acid by fed-batch Culture of Bacillus licheniformis. Biotechnol Lett, 2000, 22: 585-588
79. Zuo K, Wu W T. Semi-realtime optimization and control of a fed-batch fermentation system. Computers Chem Eng, 2000, 24: 1105-1109