姬松茸菌液体发酵及其多糖活性研究
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摘要
姬松茸是一种药食兼用真菌,其多糖具有多种生物活性。本论文以菌丝体干重产量为考察指标,通过单因素试验、Plackett-Burman试验,最陡爬坡试验和响应面法优化最佳培养基配方。并且以菌丝体干重和多糖产量为指标,优化了初始pH、种龄、接种量、装液量、转速、温度、时间等摇瓶培养条件。在单因素试验基础上,利用响应面法优化了多糖热水提取和超声提取条件。最后,研究了姬松茸菌多糖体外抗肿瘤及抗氧化活性。
Agaricus blazei Murill is a kind of fungus that was used for food and medicine. Recently, it has been used an an important nature source of immunomodulators, antimutigens and anticarcinogens. It becoming to be a promising way to develop antitumor polysaccharides, beause fungus submerged fermentation posess characteristics of low-cost and short-period. Because of the lack of wild resources and the diffculties of artificial cultivation, the submerged fermentation technology to produce medicinal fungi have a widest potential application. Some kind of medicinal fungi have been achieven the industrialization of culture in submerged fermentation. Fungal polysaccharide is one of the main active component in medicinal fungi, and has obvious effect on anti-tumor, anti-oxidation, strengthening the body immunity and anti-virus. The fungal polysaccharides research has become one of the development of new drugs. his research systematically study the liquid fermentation conditions, polysaccharide extraction process and its bioactivity in vitro.
Single factors tests were used to optimize the sort of nitrogen source, carbon source and metal ions, which were yeast extract, lactose, Na~+ and Ca~(2+). The Plackett-Burman design was investigated the effect on the dry weight of medium. Three most important factors for dry weight of medium were selected from tatal 8 factors were lactose, yeast extract, magnesium sulfate. The concentration of lactose, yeast extractive, magmesium sulfate in the medium was investigated by the means of the path of steepest ascent tests and Response Surface Methodology. The optimized values were: yeast extractive 17.13 g/L , lactose 29.34 g/L, phosphate potassium 0.3 g/L, magnesium sulfate 0.64 g/L, calcium chloride 0.01 mmol/L, sodium chloride 0.01 mmol/L, vitamin B1 100 mg/L. The maximum dry weight of mycelium of 9.56 g/L was obtained. The flask culture conditions were investigated by single factor tests. It is initial pH 5.0, inoculum age 5 days, inoculum level 7%, liquid volume 60mL, fermentation time 5 days, the mycelium dry weight 14.32 g/L, mycelium polysaccharides production 1.87g/L.
Single factor tests and Response Surface Methodology were applied to optimize the hot water extraction and ultrasound extraction conditions of Agaricus blazei Murill mycelium polysaccharides. Extracted temperature, extracted time and ratio of water to sample were chosen as the three factors of hot water extraction in RSM. The optimum hot water extraction conditions were: extracted temperature 94.7℃, extracted time 3.3 h, the ratio of water to sample 62:1. Under the optimum extraction conditions, the extraction rate of polysaccharides reach the value of 16.34%. Ultrasound extracted time, ultrasound power and the ratio of water ro sample were chosen sa the three factors of ultrasound extraction in RSM. The optimum ultrasound extraction conditions were: ultrasound extracted time 184 s, ultrasound power 410W, the ratio of water ro sample 48:1. Under the optimum extraction conditions, the extraction rate of polysaccharides reach the value of 17.44%.
MTT assay was used to study the effect of Agaricus blazei Murill polysaccharides on human tumor cells in vitro. The polysaccharides significantly inhibited the proliferation of Hep G2 and Hela in vitro. Bur the polysaccharides couldn’t inhibit the proliferation of CNE in vitro. The polysaccharides had certain reduction capacity and were able to remove a certain degree of O2-?. When clearing ? OH , the IC50 was 3.27 mg / mL,as when clearing the DPPH ? the IC50 was 5.66mg/mL.
Agaricus blazei Murill is a kind of fungus that was used for food and medicine. Recently, it has been used an an important nature source of immunomodulators, antimutigens and anticarcinogens. It becoming to be a promising way to develop antitumor polysaccharides, beause fungus submerged fermentation posess characteristics of low-cost and short-period. Because of the lack of wild resources and the diffculties of artificial cultivation, the submerged fermentation technology to produce medicinal fungi have a widest potential application. Some kind of medicinal fungi have been achieven the industrialization of culture in submerged fermentation. Fungal polysaccharide is one of the main active component in medicinal fungi, and has obvious effect on anti-tumor, anti-oxidation, strengthening the body immunity and anti-virus. The fungal polysaccharides research has become one of the development of new drugs. his research systematically study the liquid fermentation conditions, polysaccharide extraction process and its bioactivity in vitro.
Single factors tests were used to optimize the sort of nitrogen source, carbon source and metal ions, which were yeast extract, lactose, Na~+ and Ca~(2+). The Plackett-Burman design was investigated the effect on the dry weight of medium. Three most important factors for dry weight of medium were selected from tatal 8 factors were lactose, yeast extract, magnesium sulfate. The concentration of lactose, yeast extractive, magmesium sulfate in the medium was investigated by the means of the path of steepest ascent tests and Response Surface Methodology. The optimized values were: yeast extractive 17.13 g/L , lactose 29.34 g/L, phosphate potassium 0.3 g/L, magnesium sulfate 0.64 g/L, calcium chloride 0.01 mmol/L, sodium chloride 0.01 mmol/L, vitamin B1 100 mg/L. The maximum dry weight of mycelium of 9.56 g/L was obtained. The flask culture conditions were investigated by single factor tests. It is initial pH 5.0, inoculum age 5 days, inoculum level 7%, liquid volume 60mL, fermentation time 5 days, the mycelium dry weight 14.32 g/L, mycelium polysaccharides production 1.87g/L.
Single factor tests and Response Surface Methodology were applied to optimize the hot water extraction and ultrasound extraction conditions of Agaricus blazei Murill mycelium polysaccharides. Extracted temperature, extracted time and ratio of water to sample were chosen as the three factors of hot water extraction in RSM. The optimum hot water extraction conditions were: extracted temperature 94.7℃, extracted time 3.3 h, the ratio of water to sample 62:1. Under the optimum extraction conditions, the extraction rate of polysaccharides reach the value of 16.34%. Ultrasound extracted time, ultrasound power and the ratio of water ro sample were chosen sa the three factors of ultrasound extraction in RSM. The optimum ultrasound extraction conditions were: ultrasound extracted time 184 s, ultrasound power 410W, the ratio of water ro sample 48:1. Under the optimum extraction conditions, the extraction rate of polysaccharides reach the value of 17.44%.
MTT assay was used to study the effect of Agaricus blazei Murill polysaccharides on human tumor cells in vitro. The polysaccharides significantly inhibited the proliferation of Hep G2 and Hela in vitro. Bur the polysaccharides couldn’t inhibit the proliferation of CNE in vitro. The polysaccharides had certain reduction capacity and were able to remove a certain degree of O2-?. When clearing ? OH , the IC50 was 3.27 mg / mL,as when clearing the DPPH ? the IC50 was 5.66mg/mL.
引文
[1] Mizuno, Hagiwara, Nakamura, et al. A StudiesoniheHost-Mediated Antitumor Polysaceharides, Antitumor-Aetivity and Some Properties of water Soluble Polysaceharides From Hiematsutake, the Fruiting Body of Agaricus Blazei Murill[J],Agrieulture and Biologieal Chemistry, 1990, 54(11):2889-2896.
[2] Cho soo-muk, Park Jeong-Sik. Kim Kwang-Po, et al. Chemical features and purifieation of immunostimulating polysaccharides from the fruit bodies of Agaricus blaze[J],Source Korean Joumal of Myeology, 1999, 27(2):170-174
[3] Hitoshi Ito et al. Anti-tumor effect of a new polysaccharide-protein comlex prepare from Agaricus blazi“Himematsutake”and its mechanisms in tumor- bearing mice [J], Anticancer research, 1997, 17: 277-284.
[4] Bohn J, Bermillerjn,β(1-3) glucans as biological response modifiers: a review of structure functional activity relationships[J].Carbohydrate polymers, 1995, 28:314.
[5] Pirttijrvi T S M,Wahlstr m G,Rainey F A,et al. Inhibition of bacilli in industrial starches by nisin[J].Journal of Industrial Microbi-ology & Biotechnology, 2001, 26:107-114.
[6] Append in i P,Hotchkiss J H.Review of antimicrobial food packag-ing [J]. Innovative Food Science & Emerging Technologies,2002,(3):113-126.
[7] Salim A,Hassanin M A,Zohair A.A simple procedure for reducing lead content in fish [J].Food and Chemical Toxicology,2003,41:595-597.
[8] Bower C K,Parker J E,Higgins A Z,et al. Protein antimicrobial barriers to bacterial adhesion: in vitro and in vivo evaluation ofnisin-treated implantable materials[J]. Colloids and Surfaces B:Biointerfaces,2002,25:81-90.
[9] Lv W, Zhang X, CongW.Modeling the production of nisin by Lactococcus lactis[J]. Appl Microbiol Biotechnol 2005;68:322.
[10] Chuanbin Liu,Yan Liu,Wei Liao,Zhiyou Wen,et. Simultaneous Production of Nisin and Lactic Acid form Cheese Whey[J].Applide Biochemistry and Biotechnology,2004,113-116.
[11] Li WF,Wang YB,Pan YJ,et. Optimization of medium composition for nisin fermentation with response surface methodology[J].Journal of Science, 2008 ,73: M245-M249.
[12] Wenhua Lv,Wei Cong,Zhaoling Cai. Improvement of nisin production in pH feed-back controlled, fed-batch culture by Lactococcus lactis subsp. Lactis[J]. Biotechnology Letters ,2004,26: 1713-1716.
[13] Wen hua Lv, Wei Cong,Zhaoling Ca. Effect of sucrose on nisin production in batch and fed-batch culture by Lactococcus lactis[J]. Journal of Chemical Technology and Biotechnology. 2005,80:511-518.
[14] Hakim Ghalfi,Noreddine Benkerroum,et. Comparison of the performances of different fermentation strategies on cell growth and bacteriocin production by Lactobacillus curvatus CWBI-B28[J]. Journal of the Science of Food and Agriculture. 2007,87:541-549.
[15] M. Papagianni, N. Avramidis, G. Filiousis. Investigating the relationship between the specific glucose uptake rate and nisin production in aerobic batch and fed-batch glucostat cultures of Lactococcus lactis[J].Enzyme and Microbial Technology . 2007,40 :1557-1563.
[16] Nelson P.Guerra,Ana Torrado Agrasar,Cristina Lo′pez Mac?′as,et. Dynamic mathematical models to describe the growth and nisin production by Lactococcus lactis subsp. lactis CECT 539 in both batch and re-alkalized fed-batch cultures[J]. Journal of Food Engineering ,2007,82 :103-113.
[17] Hiroshi Shimizu,Taiji Mizuguchi,et.Nisin Production by a Mixed-Culture System Consisting of Lactococcus lactis and Kluyveromyces marxianus[J].Applied and Environmental Microbiology,1999,65(7):3134-3141.
[18] David W,Christianson.Five Golden Rings[J]. Science. 2006, 5766(311): 1382-1383.
[19] De Vuyst.Nutritional factors affecting nisin production by Lactococcus lactis subsp.Lactis NIZO 22186 in a synthetic medium[J].Journal of Applied Bacteriology, 1995,78:28-33
[20] Nakamura K, Yamaguchi Y, Kagota S,et al. Inhibitory effect of Cordyceps sinensis on spontaneous liver metastasis of Lewis lung carcinoma and B16 melanoma cells in syngeneic mice[J]. Jpn-J-Pharmacol, 1999, 79(3): 335-341.
[21] Knubel G, K. Larsen, E. Moore, (1990). Cytotoxic, antiviral indolocarbazoles from a blue-green alga belonging to the Nostoc. J. Antibiot. 43: 1236-1239.
[22] Cuniningham K G, Manson W, Spring F S, et al. Cordycepin, a metablic product isolated from cultures of Cordyceps Militaris Link [J]. Nature, 1950, 2; 166(4231):949.
[23] Wittek R, Koblet H, Menna A, et al. The effect of Cordycepin on the multiplication of Semliki forest virus and on polyadenylation of viralRNA [J]. Archvirol, 1977,54(1-2):95-106.
[24] Ahn YJ,Park SJ,Lee SG,et al. Cordycepin:Selective Growth Inhibitor Derived from Liquid Culture of Cordyceps Militaris Against Clostridium[J]. Agric Food Chem, 2000, 48 (7): 2744-2748.
[25] Alan M Sug ar, Ronald P Mccaffrey. Antifungal Activity of 3’2 Deoxyadenosine [J]. Antimicrobial Agents and Chemotherapy, 1998, 42(6): 1424-1427.
[26] Kim J R, Yeon SH, Kim HS, et al. Larvicidal Activity Against Plutella Xylostella of Cordycepin from the Fruiting Body of Cordyceps Militaris[J]. Pest Manag Sci, 2002, 58 (7):713-717.
[27] Bak JW, Lermer L, Chilton J, et al. Antitumor sterols from the mycelia of Cordyceps sinensis [J]. Phytochemistry, 1999, 51(7):891-898.
[28] Yang LY,Chen A,KuoYC,et al. Efficacy of a pure compound HI-A extracted from Cordyceps sinensis on autoimmune disease of MRL Ipr/Ipr mice[J]. J.Lab.Clin.Med, 1999, 134(5):492.
[29] Yoo HS, Shin JW, Cho J H, et al. Effects of Cordyceps Militaris Extract on Angiogenesis and Tumor Growth[J]. Acta Pharmacol Sin, 2004, 25(5): 657-665.
[30] Kodama EN, McCaffrey RP, Yusa K, et al. Antileukemic Activity and Mechanism of Action of Cordycepin Against Terminal Deoxynucleotidyl Transferase positive Leukemic Cells[J]. Biochem Pharmacol , 2000, 59(3): 273-281.
[31] Black PL, Henderson EE, Pfleiderer W, et al. 2’,5’-Oligoadenylate primer Core and the Cordycepin Analog Argument the Tumoricidal Activity of Human Natural Killer Cells [J]. The Journal of Immunology, 1984, 133 (5):2773-2777.
[32] Glazer RI, Lott TJ, Peale AL. Potentiation by 2’-deoxycoformycin of the Inhibitory Effect by 3’-deoxyadenosine(Cordycepin) on Nuclear RNA Synthesis in L1210 Cells in Vitro [J]. Cancer Research, 1978, 38 (8):2233-2238.
[33] Penman S, Rosbash M, and Penman M. Messenger and Heterogeneous Nuclear RNA in HeLa Cells: Differential Inhibition by Cordycepin [J]. The National Academy of Sciences, 1970, 67(4): 1878-1885.
[34] Desrosiers RC, Rottman FM, Boezi JA, et al. The Sensitivity of RNA Polymerases I and II from Novikoff Hepatoma Cells to 3’- deoxyadenosine 5’-triphosphate [J]. Nucleic Acids Research, 1976, 3 (2):325-341.
[35] Harold T Shigeura, Charles N Gordon. The Effects of 3’-Deoxyadenosine on the Synthesis of Ribonucleic Acid[J]. The Journal of Biological Chemistry, 1965, 240 (2): 806-810.
[36] Ioannidis P, Courtis N, Havredaki M, et al. The Polyadenylation Inhibitor Cordycepin Cause a Decline in c-MYC mRNA Levels Without Effecting c-MYC Protein Levels[J]. Oncogene, 1999, 18 (1): 117-125.
[37] Moshe Siev, Robert Weinberg, Sheldon Penman. The Selective Interruption of Nucleolar RNA Synthesis in the Hela Cells by Cordycepin [J]. The Journal of Cell Biology, 1969, 41, 510-520.
[38] Mendecki J, Lee SY, Brawerman G. Characteristics of the Polyadenylic Acid Segment Associated with Messenger Ribonucleic Acid in Mouse Sarcoma 180 Ascites Cells[J]. Biochemistry, 1972, 11(5):792-798.
[39] Koc Y, Urbano AG, Sweeney EB, et al. Induction of Apoptosis by Cordycepin in ADA-inhibited TdT - positiveLeukemia Cells [J]. Leukemia, 1996, 10(6): 1019-1024.
[40] Fuller BB, Lunsford JB, Iman DS. Alpha-melanocyte-stimulating Hormone Regulation of Tyrosinase in Cloudman S-91 Mouse Melanoma Cell Cultures[J]. Biol Chem, 1987, 262(9): 4024-4033.
[41] Li SP, Zhao KJ, Ji ZN, et al. A Polysaccharide Isolated from Cordyceps Sinensis , a Traditional Chinese Medicine ,Protects PC12 Cells Against Hydrogen Peroxide-induced Injury [J]. Life Sci, 2003, 73(19): 2503-2513.
[42] Majone F, Montaldi A, Ronchese F, et al. Cordycepin Reduces the Sensitivity of BALB Mo Mouse Lymphocytes to the Induction of Sister Chromatid Exchanges [J].Carcinogenesis, 1985, 6:131-134.
[43] Majone F, Montaldi A, Saggioro D, et al. Differential Effects of Cordycepin on the Induction of Sister-chromatid Exchange and Chromatid Breaks in BALB/ Mo Mouse Lymphocytes Treated with Mitomycin C [J]. Mutagenesis, 1987, 2:259-262.
[44] Richardson LS, Ting RC, Gallo RC,et al. Effect of Cordycepin on the Replication of type-c RNA Tumor Viruses[J]. Int J Cancer, 1975, 15(3): 451-456.
[45] Roa RC, Subir K. Bose. Requirement for Cellular Protein Synthesis in Reversal of Ethidiumbromide-Induced Inhibition of Cell Transformation by Murine Sarcoma Virus[J]. PNAS, 1975, 72(11):4337-4340.
[46] Rombouts F M, Thibault J. Feruloylated pectic substances from sugar- beet pulp [J].Carbohydrate Research, 1986, 154:177-187.
[47] Sun R,Hughes S.Fractional extraction and physico-chemical characterization of hemicelluloses and cellulose from sugar beet pulp[J].Carbohydrate Polymers, 1998, 36:293-299.
[48] Ring S G, Selvendran R R.An arabinogalactoxyloglucan from the cell wall of Solanum tuberosum [J]. Phytochemistry, 1981, 20(11): 2511-2519.
[49] Tsaffrir Zor, Zvi Selinger, linearization of the Bradford protein assay increases its sensitivity:theoretical and experimental studies, Analytical Biochemistry, 1996, 236:302-308.
[50] Blumenkrantz N,Asboe-Hansen G.New method for quantitative determination of uronic acids.[J]. Analytical Biochemistry, 1973, 54:484-489.
[51] Miyazaki T, et al.Studies on fungal polysaccharides XX. Galactomannan of Cordyceps sinensis[J].ChemPharmBull, 1977, 25(12):3324-3328.
[52] Yang B.,K, Ha J., Y., Jeong S., et al. Production of exo-polymers by submerged mycelial culture of Cordyceps militaris and its hypolipidemic effect. J Microbiol Biotechn. 2000, 10(6): 784-788.
[53] Xu C., P., Kim S., W., Hwang H., J., et al. Optimization of submerged culture conditions for mycelial growth and exo-biopolymer production by Paecilomyces tenuipes C240. Process Biochem. 2003, 38(7): 1025-1030.
[54] Xu C., P., Yun J., W. Optimization of submerged culture conditions for mycelial growth and exo-biopolymer production by Auricularia polytricha using the methods of uniform design and regression analysis. Biotechnol Appl Bioc. 2003, 38: 193-199.
[55] Lim J., M., Kim S.,W., Hwang H., J., et al. Optimization of medium by orthogonal matrix method for submerged mycelial culture and exopolysaccharides production in Collybia maculata. Appl Biochem Biotech. 2004, 119(2): 159-170.
[56] Kim H., O., Lim J., M., Joo J., H., et al. Optimization of submerged culture conditions for the production of mycelial biomass and exopolysaccharide production by Agrocybe cylindracea. BioresTechn. 2005, 96: 1175-1182.
[57] Wu J., Z., Cheung P., C., K., Wonga K., H., et al. Studies on submerged fermentation of Pleurotus tuber-regium (Fr.) Singer-Part 1: physical and chemical factors affecting the rate of mycelial growth and bioconversion efficiency. Food Chem. 2003, 81(3): 389-393.
[58] Wu J., Z., Cheung P., C., K., Wonga K., H., et al. Studies on submerged fermentation of Pleurotus tuber-regium (Fr.) Singer. Part 2: effect of carbon-to-nitrogen ratio of the culture medium on the content and composition of the mycelial dietary fibre. Food Chem. 2004, 85(1): 101-105.
[59] Fang Q., H., Zhong J., J. Submerged fermentation of higher fungus Ganoderma lucidum for production of valuable bioactive metabolites-ganoderic acid and polysaccharide. Biochem Eng J. 2002, 10(1): 61-65.
[60] Yang H., L., Wu T., X., Zhang K., C. Enhancement production of mycelial growth and in Ganoderma lucidum (the Chinese medical fungus,lingzhi) by polysaccharide the addition of ethanol. Biotechn Lett. 2004, 26: 841-844.
[61] Fang F., C., Liau C., B. The influence of environmental conditions on polysaccharide formation by Gavoderma lucidium in submerged cultures. Process Biochem. 1998, 33(5): 547-553.
[62] Fang Q., H., Zhong J., J. Effect of initial pH on production of ganoderic acid and polysaccharide by submerged fermentation of Ganoderma lucidum. Process Biochem. 2002, 37(7): 769-774.
[63] Tang Y., J., Zhong J., J. Role of oxygen supply in submerged fermentation of Ganoderma lucidum for production of Ganoderma polysaccharide and ganoderic acid. Enzyme Microb Tech. 2003, 32(3): 478-484.
[64] Kim S., W., Hwang H., J., Xu C., P., et al. Effect of aeration and agitation on the production of mycelial biomass and exopolysaccharides in an enthomopathogenic fungus Paecilomyces sinclairii. Lett Appl Microb. 2003, 36: 321-326.
[65] Xu C., P., Yun J., W. Influence of aeration on the production and the quality of the exopolysaccharides from Paecilomyces tenuipes C240 in a stirred-tank fermenter. Enzyme Microb Tech. 2004, 35: 33-39.
[66] Ohno N, lino K,Suzuki I, et al .Neutral and acidic antitumor polysaccharides Extractedn from cultured fruit bodies of grifola frondosa. Chem. Pharm. Bull., 1985, 33(3): 1181-1186.
[67] Xiang DB, Li XY. Antitumor activity and immuno-potentiating actions of Achyanthes bidentata polysaccharides.Acta Pharmacol Sin, 1993, 14: 556-56.
[68] Jie Z, Guan YW, Hang L,et al.Antitumor active protein-containing Glycans from the Chinese mushroom Songshan Lingzhi,Ganoderma tsuge mycelium. Biosci Biotech Biochem, 1994, 158: 1202-120.
[69] Lsdanyi A.Effect of Lentinan on marcrophage cytotoxicity againstmetastatic tumor cells , cancer immunol.Immuorner, 1993, 36(4): 123-12.
[70] Ohno N, Suzuke I, Oikawa S, et al.Antitumor activity and structural characterization of Glucans extracted from cultured fruit bodies ofGrifola frondosa. Chemical and Pharmaceutical Bulletin, 1984, 32: 1142-1151.
[71] Nanba H, Hamaguchi A, Kuroda H. The chemical structure of an antitumor polysaccharide in fruit bodies ofGrifola frondosa (Maitake).Chemical and Pharmaceutical Bulletin, 1987, 35: 1162-1168.
[72] Solomon P. Wasser, The Extraction and Development of Antitumor-Active Polysaccharides Mushrooms in Japan(Review), International J of Medicinal Mushroom, 1999, 1:44.
[73] Robert A, Stowe R, Mayer P.Efficient screening of process variables. Industrial and Engineering Chemistry, 1966, 58(2): 36-40.
[74] Paula M L, Castro P M, Hayter A, Ison P.Application of a statistical design to the optimization of culture medium for recombinant interferon-gamma production by Chinese hamster ovary cells. Appl. Microbiol. Biotechnol., 1992, 38:84-90.
[75] Hounsa C G., Aubry J M, Dubourguier H C.Application of factorial and Doehlert designs for optimization of pectate lyase production by a recombinant Escherichia coli.Appl. Microbiol. Biotechnol., 1996, 45: 764-770.
[76] Rao K J, Kim C H, Rhee S K. Statistical optimization of medium for the production of recombinant hirudin from Saccharomyces cerevisiae using response surface methodology. Process Biochemistry, 2000, 35: 639-647.
[77] Rajiv V, Pranav V, Chhatpar H S. Statistical optimization of medium components for the production of chitinase by Alcaligenes xylosoxydans. Enzyme and Microbiol Technology, 2003, 33, 92-96.
[78] Martin J F, Demain A L. Organization and expression of genes involved in the biosynthesis of antibiotics and other secondary metabolites. Annual Review of Microbiology, 1989, 43, 173-206.
[79] Feng Y., Y., He Z., M., Ong S., L., et al. Optimization of agitation, aeration, and temperature conditions for maximumβ-mannanase production. Enzyme Microb Tech. 2003, 32: 282-289.
[80] Maria Papagianni. Fungal morphology and metabolite production in submerged mycelial processes[J]. Biotechnology Advances 22 (2004) 189-259.
[81] L. de Mare′C. CimanderA. Elfwing P. Hagander. Feeding strategies for E. coli fermentations demanding an enriched environment[J]. Bioprocess Biosyst Eng (2007) 30:13-25.
[82] G. L. Riley, K. G. Tucker,etc. Effect of Biomass Concentration and Mycelial Morphology on Fermentation Broth Rheology[J]. BIOTECHNOLOGY AND BIOENGINEERING, VOL. 68, NO. 2, APRIL 20, 2000.
[83] N.T.P. Dung, F.M. Rombouts,etc. Functionality of selected strains of moulds and yeasts from Vietnamese rice wine starters [J]. Food Microbiology 23 (2006) 331-340.
[84] M. Papagianni, M. Mattey, B. Kristiansen. The influence of glucose concentration on citric acid production and morphology of Aspergillus niger in batch and culture[J]. Enzyme and Microbial Technology 25 (1999) 710-717.
[2] Cho soo-muk, Park Jeong-Sik. Kim Kwang-Po, et al. Chemical features and purifieation of immunostimulating polysaccharides from the fruit bodies of Agaricus blaze[J],Source Korean Joumal of Myeology, 1999, 27(2):170-174
[3] Hitoshi Ito et al. Anti-tumor effect of a new polysaccharide-protein comlex prepare from Agaricus blazi“Himematsutake”and its mechanisms in tumor- bearing mice [J], Anticancer research, 1997, 17: 277-284.
[4] Bohn J, Bermillerjn,β(1-3) glucans as biological response modifiers: a review of structure functional activity relationships[J].Carbohydrate polymers, 1995, 28:314.
[5] Pirttijrvi T S M,Wahlstr m G,Rainey F A,et al. Inhibition of bacilli in industrial starches by nisin[J].Journal of Industrial Microbi-ology & Biotechnology, 2001, 26:107-114.
[6] Append in i P,Hotchkiss J H.Review of antimicrobial food packag-ing [J]. Innovative Food Science & Emerging Technologies,2002,(3):113-126.
[7] Salim A,Hassanin M A,Zohair A.A simple procedure for reducing lead content in fish [J].Food and Chemical Toxicology,2003,41:595-597.
[8] Bower C K,Parker J E,Higgins A Z,et al. Protein antimicrobial barriers to bacterial adhesion: in vitro and in vivo evaluation ofnisin-treated implantable materials[J]. Colloids and Surfaces B:Biointerfaces,2002,25:81-90.
[9] Lv W, Zhang X, CongW.Modeling the production of nisin by Lactococcus lactis[J]. Appl Microbiol Biotechnol 2005;68:322.
[10] Chuanbin Liu,Yan Liu,Wei Liao,Zhiyou Wen,et. Simultaneous Production of Nisin and Lactic Acid form Cheese Whey[J].Applide Biochemistry and Biotechnology,2004,113-116.
[11] Li WF,Wang YB,Pan YJ,et. Optimization of medium composition for nisin fermentation with response surface methodology[J].Journal of Science, 2008 ,73: M245-M249.
[12] Wenhua Lv,Wei Cong,Zhaoling Cai. Improvement of nisin production in pH feed-back controlled, fed-batch culture by Lactococcus lactis subsp. Lactis[J]. Biotechnology Letters ,2004,26: 1713-1716.
[13] Wen hua Lv, Wei Cong,Zhaoling Ca. Effect of sucrose on nisin production in batch and fed-batch culture by Lactococcus lactis[J]. Journal of Chemical Technology and Biotechnology. 2005,80:511-518.
[14] Hakim Ghalfi,Noreddine Benkerroum,et. Comparison of the performances of different fermentation strategies on cell growth and bacteriocin production by Lactobacillus curvatus CWBI-B28[J]. Journal of the Science of Food and Agriculture. 2007,87:541-549.
[15] M. Papagianni, N. Avramidis, G. Filiousis. Investigating the relationship between the specific glucose uptake rate and nisin production in aerobic batch and fed-batch glucostat cultures of Lactococcus lactis[J].Enzyme and Microbial Technology . 2007,40 :1557-1563.
[16] Nelson P.Guerra,Ana Torrado Agrasar,Cristina Lo′pez Mac?′as,et. Dynamic mathematical models to describe the growth and nisin production by Lactococcus lactis subsp. lactis CECT 539 in both batch and re-alkalized fed-batch cultures[J]. Journal of Food Engineering ,2007,82 :103-113.
[17] Hiroshi Shimizu,Taiji Mizuguchi,et.Nisin Production by a Mixed-Culture System Consisting of Lactococcus lactis and Kluyveromyces marxianus[J].Applied and Environmental Microbiology,1999,65(7):3134-3141.
[18] David W,Christianson.Five Golden Rings[J]. Science. 2006, 5766(311): 1382-1383.
[19] De Vuyst.Nutritional factors affecting nisin production by Lactococcus lactis subsp.Lactis NIZO 22186 in a synthetic medium[J].Journal of Applied Bacteriology, 1995,78:28-33
[20] Nakamura K, Yamaguchi Y, Kagota S,et al. Inhibitory effect of Cordyceps sinensis on spontaneous liver metastasis of Lewis lung carcinoma and B16 melanoma cells in syngeneic mice[J]. Jpn-J-Pharmacol, 1999, 79(3): 335-341.
[21] Knubel G, K. Larsen, E. Moore, (1990). Cytotoxic, antiviral indolocarbazoles from a blue-green alga belonging to the Nostoc. J. Antibiot. 43: 1236-1239.
[22] Cuniningham K G, Manson W, Spring F S, et al. Cordycepin, a metablic product isolated from cultures of Cordyceps Militaris Link [J]. Nature, 1950, 2; 166(4231):949.
[23] Wittek R, Koblet H, Menna A, et al. The effect of Cordycepin on the multiplication of Semliki forest virus and on polyadenylation of viralRNA [J]. Archvirol, 1977,54(1-2):95-106.
[24] Ahn YJ,Park SJ,Lee SG,et al. Cordycepin:Selective Growth Inhibitor Derived from Liquid Culture of Cordyceps Militaris Against Clostridium[J]. Agric Food Chem, 2000, 48 (7): 2744-2748.
[25] Alan M Sug ar, Ronald P Mccaffrey. Antifungal Activity of 3’2 Deoxyadenosine [J]. Antimicrobial Agents and Chemotherapy, 1998, 42(6): 1424-1427.
[26] Kim J R, Yeon SH, Kim HS, et al. Larvicidal Activity Against Plutella Xylostella of Cordycepin from the Fruiting Body of Cordyceps Militaris[J]. Pest Manag Sci, 2002, 58 (7):713-717.
[27] Bak JW, Lermer L, Chilton J, et al. Antitumor sterols from the mycelia of Cordyceps sinensis [J]. Phytochemistry, 1999, 51(7):891-898.
[28] Yang LY,Chen A,KuoYC,et al. Efficacy of a pure compound HI-A extracted from Cordyceps sinensis on autoimmune disease of MRL Ipr/Ipr mice[J]. J.Lab.Clin.Med, 1999, 134(5):492.
[29] Yoo HS, Shin JW, Cho J H, et al. Effects of Cordyceps Militaris Extract on Angiogenesis and Tumor Growth[J]. Acta Pharmacol Sin, 2004, 25(5): 657-665.
[30] Kodama EN, McCaffrey RP, Yusa K, et al. Antileukemic Activity and Mechanism of Action of Cordycepin Against Terminal Deoxynucleotidyl Transferase positive Leukemic Cells[J]. Biochem Pharmacol , 2000, 59(3): 273-281.
[31] Black PL, Henderson EE, Pfleiderer W, et al. 2’,5’-Oligoadenylate primer Core and the Cordycepin Analog Argument the Tumoricidal Activity of Human Natural Killer Cells [J]. The Journal of Immunology, 1984, 133 (5):2773-2777.
[32] Glazer RI, Lott TJ, Peale AL. Potentiation by 2’-deoxycoformycin of the Inhibitory Effect by 3’-deoxyadenosine(Cordycepin) on Nuclear RNA Synthesis in L1210 Cells in Vitro [J]. Cancer Research, 1978, 38 (8):2233-2238.
[33] Penman S, Rosbash M, and Penman M. Messenger and Heterogeneous Nuclear RNA in HeLa Cells: Differential Inhibition by Cordycepin [J]. The National Academy of Sciences, 1970, 67(4): 1878-1885.
[34] Desrosiers RC, Rottman FM, Boezi JA, et al. The Sensitivity of RNA Polymerases I and II from Novikoff Hepatoma Cells to 3’- deoxyadenosine 5’-triphosphate [J]. Nucleic Acids Research, 1976, 3 (2):325-341.
[35] Harold T Shigeura, Charles N Gordon. The Effects of 3’-Deoxyadenosine on the Synthesis of Ribonucleic Acid[J]. The Journal of Biological Chemistry, 1965, 240 (2): 806-810.
[36] Ioannidis P, Courtis N, Havredaki M, et al. The Polyadenylation Inhibitor Cordycepin Cause a Decline in c-MYC mRNA Levels Without Effecting c-MYC Protein Levels[J]. Oncogene, 1999, 18 (1): 117-125.
[37] Moshe Siev, Robert Weinberg, Sheldon Penman. The Selective Interruption of Nucleolar RNA Synthesis in the Hela Cells by Cordycepin [J]. The Journal of Cell Biology, 1969, 41, 510-520.
[38] Mendecki J, Lee SY, Brawerman G. Characteristics of the Polyadenylic Acid Segment Associated with Messenger Ribonucleic Acid in Mouse Sarcoma 180 Ascites Cells[J]. Biochemistry, 1972, 11(5):792-798.
[39] Koc Y, Urbano AG, Sweeney EB, et al. Induction of Apoptosis by Cordycepin in ADA-inhibited TdT - positiveLeukemia Cells [J]. Leukemia, 1996, 10(6): 1019-1024.
[40] Fuller BB, Lunsford JB, Iman DS. Alpha-melanocyte-stimulating Hormone Regulation of Tyrosinase in Cloudman S-91 Mouse Melanoma Cell Cultures[J]. Biol Chem, 1987, 262(9): 4024-4033.
[41] Li SP, Zhao KJ, Ji ZN, et al. A Polysaccharide Isolated from Cordyceps Sinensis , a Traditional Chinese Medicine ,Protects PC12 Cells Against Hydrogen Peroxide-induced Injury [J]. Life Sci, 2003, 73(19): 2503-2513.
[42] Majone F, Montaldi A, Ronchese F, et al. Cordycepin Reduces the Sensitivity of BALB Mo Mouse Lymphocytes to the Induction of Sister Chromatid Exchanges [J].Carcinogenesis, 1985, 6:131-134.
[43] Majone F, Montaldi A, Saggioro D, et al. Differential Effects of Cordycepin on the Induction of Sister-chromatid Exchange and Chromatid Breaks in BALB/ Mo Mouse Lymphocytes Treated with Mitomycin C [J]. Mutagenesis, 1987, 2:259-262.
[44] Richardson LS, Ting RC, Gallo RC,et al. Effect of Cordycepin on the Replication of type-c RNA Tumor Viruses[J]. Int J Cancer, 1975, 15(3): 451-456.
[45] Roa RC, Subir K. Bose. Requirement for Cellular Protein Synthesis in Reversal of Ethidiumbromide-Induced Inhibition of Cell Transformation by Murine Sarcoma Virus[J]. PNAS, 1975, 72(11):4337-4340.
[46] Rombouts F M, Thibault J. Feruloylated pectic substances from sugar- beet pulp [J].Carbohydrate Research, 1986, 154:177-187.
[47] Sun R,Hughes S.Fractional extraction and physico-chemical characterization of hemicelluloses and cellulose from sugar beet pulp[J].Carbohydrate Polymers, 1998, 36:293-299.
[48] Ring S G, Selvendran R R.An arabinogalactoxyloglucan from the cell wall of Solanum tuberosum [J]. Phytochemistry, 1981, 20(11): 2511-2519.
[49] Tsaffrir Zor, Zvi Selinger, linearization of the Bradford protein assay increases its sensitivity:theoretical and experimental studies, Analytical Biochemistry, 1996, 236:302-308.
[50] Blumenkrantz N,Asboe-Hansen G.New method for quantitative determination of uronic acids.[J]. Analytical Biochemistry, 1973, 54:484-489.
[51] Miyazaki T, et al.Studies on fungal polysaccharides XX. Galactomannan of Cordyceps sinensis[J].ChemPharmBull, 1977, 25(12):3324-3328.
[52] Yang B.,K, Ha J., Y., Jeong S., et al. Production of exo-polymers by submerged mycelial culture of Cordyceps militaris and its hypolipidemic effect. J Microbiol Biotechn. 2000, 10(6): 784-788.
[53] Xu C., P., Kim S., W., Hwang H., J., et al. Optimization of submerged culture conditions for mycelial growth and exo-biopolymer production by Paecilomyces tenuipes C240. Process Biochem. 2003, 38(7): 1025-1030.
[54] Xu C., P., Yun J., W. Optimization of submerged culture conditions for mycelial growth and exo-biopolymer production by Auricularia polytricha using the methods of uniform design and regression analysis. Biotechnol Appl Bioc. 2003, 38: 193-199.
[55] Lim J., M., Kim S.,W., Hwang H., J., et al. Optimization of medium by orthogonal matrix method for submerged mycelial culture and exopolysaccharides production in Collybia maculata. Appl Biochem Biotech. 2004, 119(2): 159-170.
[56] Kim H., O., Lim J., M., Joo J., H., et al. Optimization of submerged culture conditions for the production of mycelial biomass and exopolysaccharide production by Agrocybe cylindracea. BioresTechn. 2005, 96: 1175-1182.
[57] Wu J., Z., Cheung P., C., K., Wonga K., H., et al. Studies on submerged fermentation of Pleurotus tuber-regium (Fr.) Singer-Part 1: physical and chemical factors affecting the rate of mycelial growth and bioconversion efficiency. Food Chem. 2003, 81(3): 389-393.
[58] Wu J., Z., Cheung P., C., K., Wonga K., H., et al. Studies on submerged fermentation of Pleurotus tuber-regium (Fr.) Singer. Part 2: effect of carbon-to-nitrogen ratio of the culture medium on the content and composition of the mycelial dietary fibre. Food Chem. 2004, 85(1): 101-105.
[59] Fang Q., H., Zhong J., J. Submerged fermentation of higher fungus Ganoderma lucidum for production of valuable bioactive metabolites-ganoderic acid and polysaccharide. Biochem Eng J. 2002, 10(1): 61-65.
[60] Yang H., L., Wu T., X., Zhang K., C. Enhancement production of mycelial growth and in Ganoderma lucidum (the Chinese medical fungus,lingzhi) by polysaccharide the addition of ethanol. Biotechn Lett. 2004, 26: 841-844.
[61] Fang F., C., Liau C., B. The influence of environmental conditions on polysaccharide formation by Gavoderma lucidium in submerged cultures. Process Biochem. 1998, 33(5): 547-553.
[62] Fang Q., H., Zhong J., J. Effect of initial pH on production of ganoderic acid and polysaccharide by submerged fermentation of Ganoderma lucidum. Process Biochem. 2002, 37(7): 769-774.
[63] Tang Y., J., Zhong J., J. Role of oxygen supply in submerged fermentation of Ganoderma lucidum for production of Ganoderma polysaccharide and ganoderic acid. Enzyme Microb Tech. 2003, 32(3): 478-484.
[64] Kim S., W., Hwang H., J., Xu C., P., et al. Effect of aeration and agitation on the production of mycelial biomass and exopolysaccharides in an enthomopathogenic fungus Paecilomyces sinclairii. Lett Appl Microb. 2003, 36: 321-326.
[65] Xu C., P., Yun J., W. Influence of aeration on the production and the quality of the exopolysaccharides from Paecilomyces tenuipes C240 in a stirred-tank fermenter. Enzyme Microb Tech. 2004, 35: 33-39.
[66] Ohno N, lino K,Suzuki I, et al .Neutral and acidic antitumor polysaccharides Extractedn from cultured fruit bodies of grifola frondosa. Chem. Pharm. Bull., 1985, 33(3): 1181-1186.
[67] Xiang DB, Li XY. Antitumor activity and immuno-potentiating actions of Achyanthes bidentata polysaccharides.Acta Pharmacol Sin, 1993, 14: 556-56.
[68] Jie Z, Guan YW, Hang L,et al.Antitumor active protein-containing Glycans from the Chinese mushroom Songshan Lingzhi,Ganoderma tsuge mycelium. Biosci Biotech Biochem, 1994, 158: 1202-120.
[69] Lsdanyi A.Effect of Lentinan on marcrophage cytotoxicity againstmetastatic tumor cells , cancer immunol.Immuorner, 1993, 36(4): 123-12.
[70] Ohno N, Suzuke I, Oikawa S, et al.Antitumor activity and structural characterization of Glucans extracted from cultured fruit bodies ofGrifola frondosa. Chemical and Pharmaceutical Bulletin, 1984, 32: 1142-1151.
[71] Nanba H, Hamaguchi A, Kuroda H. The chemical structure of an antitumor polysaccharide in fruit bodies ofGrifola frondosa (Maitake).Chemical and Pharmaceutical Bulletin, 1987, 35: 1162-1168.
[72] Solomon P. Wasser, The Extraction and Development of Antitumor-Active Polysaccharides Mushrooms in Japan(Review), International J of Medicinal Mushroom, 1999, 1:44.
[73] Robert A, Stowe R, Mayer P.Efficient screening of process variables. Industrial and Engineering Chemistry, 1966, 58(2): 36-40.
[74] Paula M L, Castro P M, Hayter A, Ison P.Application of a statistical design to the optimization of culture medium for recombinant interferon-gamma production by Chinese hamster ovary cells. Appl. Microbiol. Biotechnol., 1992, 38:84-90.
[75] Hounsa C G., Aubry J M, Dubourguier H C.Application of factorial and Doehlert designs for optimization of pectate lyase production by a recombinant Escherichia coli.Appl. Microbiol. Biotechnol., 1996, 45: 764-770.
[76] Rao K J, Kim C H, Rhee S K. Statistical optimization of medium for the production of recombinant hirudin from Saccharomyces cerevisiae using response surface methodology. Process Biochemistry, 2000, 35: 639-647.
[77] Rajiv V, Pranav V, Chhatpar H S. Statistical optimization of medium components for the production of chitinase by Alcaligenes xylosoxydans. Enzyme and Microbiol Technology, 2003, 33, 92-96.
[78] Martin J F, Demain A L. Organization and expression of genes involved in the biosynthesis of antibiotics and other secondary metabolites. Annual Review of Microbiology, 1989, 43, 173-206.
[79] Feng Y., Y., He Z., M., Ong S., L., et al. Optimization of agitation, aeration, and temperature conditions for maximumβ-mannanase production. Enzyme Microb Tech. 2003, 32: 282-289.
[80] Maria Papagianni. Fungal morphology and metabolite production in submerged mycelial processes[J]. Biotechnology Advances 22 (2004) 189-259.
[81] L. de Mare′C. CimanderA. Elfwing P. Hagander. Feeding strategies for E. coli fermentations demanding an enriched environment[J]. Bioprocess Biosyst Eng (2007) 30:13-25.
[82] G. L. Riley, K. G. Tucker,etc. Effect of Biomass Concentration and Mycelial Morphology on Fermentation Broth Rheology[J]. BIOTECHNOLOGY AND BIOENGINEERING, VOL. 68, NO. 2, APRIL 20, 2000.
[83] N.T.P. Dung, F.M. Rombouts,etc. Functionality of selected strains of moulds and yeasts from Vietnamese rice wine starters [J]. Food Microbiology 23 (2006) 331-340.
[84] M. Papagianni, M. Mattey, B. Kristiansen. The influence of glucose concentration on citric acid production and morphology of Aspergillus niger in batch and culture[J]. Enzyme and Microbial Technology 25 (1999) 710-717.