农用抗寄生虫抗生素尼莫克汀发酵工艺的初步研究
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摘要
为优化蓝灰链霉菌产尼莫克汀的发酵工艺。通过摇瓶法对其发酵条件进行了初步研究,采用单因素分析法,考察了时间、温度、溶氧及接种量对蓝灰链霉菌发酵产尼莫克汀的影响。结果表明:菌龄为8天的蓝灰链霉菌孢子适用于制种,并以甘油管法低温保存建成工作细胞库,但保存时间应不超过2个月;将工作细胞库中的菌种接入种子培养基中,置于28℃培养28 h,种子质量较好。摇瓶发酵的较优条件是容量为250 mL的三角瓶中装有发酵培养基50 mL,以5%的比例加入培养好的种子液,将发酵瓶置于25℃以250 r/min的转速发酵8天,尼莫克汀产量可达164.90μg/mL。
In order to optimize the fermentation process of nemadectin produced by Streptomyces cyaneogriseus ssp. Noncyanogenus, the fermentation conditions was primarily studied using the method of shaking flask. The effects of some factors, such as time, temperature, dissolved oxygen and inoculation volume, were demonstrated through single factor tests. The results showed that spores of S. cyaneogriseus ssp. noncyanogenus incubated for 8 d were applicable to seed preparation, and then the strain's working cell bank was established by glycerol tube cryopreservation. But the save time should be no more than 2 months. While the strain in the working cell bank was inoculated into seed medium and then cultured at 28℃ for 28 h, the seed of better quality was obtained. The optimum fermentation condition of shaking flask was that the strain seed culture was inoculated into 50 mL of fermentation medium in the flask of 250 mL with the ratio of 5% and fermented at 25℃ for 8 d with shaking speed of 250r/min. It indicated that nemadectin yield was up to 164.90μg/mL.
In order to optimize the fermentation process of nemadectin produced by Streptomyces cyaneogriseus ssp. Noncyanogenus, the fermentation conditions was primarily studied using the method of shaking flask. The effects of some factors, such as time, temperature, dissolved oxygen and inoculation volume, were demonstrated through single factor tests. The results showed that spores of S. cyaneogriseus ssp. noncyanogenus incubated for 8 d were applicable to seed preparation, and then the strain's working cell bank was established by glycerol tube cryopreservation. But the save time should be no more than 2 months. While the strain in the working cell bank was inoculated into seed medium and then cultured at 28℃ for 28 h, the seed of better quality was obtained. The optimum fermentation condition of shaking flask was that the strain seed culture was inoculated into 50 mL of fermentation medium in the flask of 250 mL with the ratio of 5% and fermented at 25℃ for 8 d with shaking speed of 250r/min. It indicated that nemadectin yield was up to 164.90μg/mL.
引文
[1]邱德文.生物农药研究进展与未来展望[J].植物保护,2013,39(5):81-89.
[2]Carter G T,Nietsche J A,Borders D B.Strucure determination of LL-F28249α,β,γandλ,potent antiparasitic macrolides from Streptomyces cyaneogriseus ssp.noncyanogenus[J].Journal of the Chemical Society-Chemical Communications,1987,(6):402-404.
[3]Doscher M E,Wood I B,Pankavich J A,et al.Efficacy of nemadectin,a new broad-spectrum endectocide,against natural infections of canine gastrointestinal helminths[J].Veterinary Parasitology,1989,34(3):255-259.
[4]Lyons E T,Tolliver S C,Drudge J H,et al.Critical and controlled tests of activity of moxidectin(CL 301,423)against natural infections of internal parasites of equids[J].Veterinary Parasitology,1992,41(3-4):255-284.
[5]Davies H G,Green R H.Avermectins and milbemycins[J].Nat Prod Rep,1986,3:87-121.
[6]Shoop W L,Mrozik H,Fisher M H.Structure and activity of avermectins and milbemycins in animal health[J].Vet Parasitol,1995,59:139-156.
[7]Roger Prichard,Cécile Ménez,Anne Lespine,et al.Moxidectin and the avermectins:Consanguinity but not identity[J].International Journal for Parasitology:Drugs and Drug Resistance,2012,2:134-153.
[8]胡萍.链霉菌neau3次级代谢产物的提取、分离及结构鉴定[D].哈尔滨:东北农业大学,2010:14-57.
[9]Yang L Y,Wang J D,Zhang J,et al.New nemadectin congeners with acaricidal and nematocidal activity from Streptomyces microflavus neau3 Y-3[J].Bioorganic&Medicinal Chemistry Letters,2013,23(20):5710-5713.
[10]王则.农用抗生素尼莫克汀高产菌株的推理选育[D].哈尔滨:东北农业大学,2009:8-52.
[11]骆健美,金志华,岑沛霖.培养条件对褐黄孢链霉菌发酵合成纳他霉素的影响[J].浙江大学学报:工学版,2005,39(2):296-300.
[12]Braun S,Vecht-lifshitz S E.Mycelial morphology and metabolite production[J].Trends Biotechnol,1991,9(2):63-68.
[13]Znidarisic P,Komel R,Pavko A.Studies of a pelleted growth form of Rhizopus nigricans as a biocatalyst for progesterone 11α-hydroxylation[J].J Biotechnology,1991,60(3):207-216.
[14]Tucker K G,Thomas C R.Inoculum effects on fungal morphology:Shake flask vs.agitated bioreactor[J].Biotechnology Techniques,1994,8(3):153-156.
[15]Shoop W L,Morzik H,Fisher M H.Structure and activity of avermectins and nemadectin in animal health[J].Vet Parasitol,1995,59:139-151.
[2]Carter G T,Nietsche J A,Borders D B.Strucure determination of LL-F28249α,β,γandλ,potent antiparasitic macrolides from Streptomyces cyaneogriseus ssp.noncyanogenus[J].Journal of the Chemical Society-Chemical Communications,1987,(6):402-404.
[3]Doscher M E,Wood I B,Pankavich J A,et al.Efficacy of nemadectin,a new broad-spectrum endectocide,against natural infections of canine gastrointestinal helminths[J].Veterinary Parasitology,1989,34(3):255-259.
[4]Lyons E T,Tolliver S C,Drudge J H,et al.Critical and controlled tests of activity of moxidectin(CL 301,423)against natural infections of internal parasites of equids[J].Veterinary Parasitology,1992,41(3-4):255-284.
[5]Davies H G,Green R H.Avermectins and milbemycins[J].Nat Prod Rep,1986,3:87-121.
[6]Shoop W L,Mrozik H,Fisher M H.Structure and activity of avermectins and milbemycins in animal health[J].Vet Parasitol,1995,59:139-156.
[7]Roger Prichard,Cécile Ménez,Anne Lespine,et al.Moxidectin and the avermectins:Consanguinity but not identity[J].International Journal for Parasitology:Drugs and Drug Resistance,2012,2:134-153.
[8]胡萍.链霉菌neau3次级代谢产物的提取、分离及结构鉴定[D].哈尔滨:东北农业大学,2010:14-57.
[9]Yang L Y,Wang J D,Zhang J,et al.New nemadectin congeners with acaricidal and nematocidal activity from Streptomyces microflavus neau3 Y-3[J].Bioorganic&Medicinal Chemistry Letters,2013,23(20):5710-5713.
[10]王则.农用抗生素尼莫克汀高产菌株的推理选育[D].哈尔滨:东北农业大学,2009:8-52.
[11]骆健美,金志华,岑沛霖.培养条件对褐黄孢链霉菌发酵合成纳他霉素的影响[J].浙江大学学报:工学版,2005,39(2):296-300.
[12]Braun S,Vecht-lifshitz S E.Mycelial morphology and metabolite production[J].Trends Biotechnol,1991,9(2):63-68.
[13]Znidarisic P,Komel R,Pavko A.Studies of a pelleted growth form of Rhizopus nigricans as a biocatalyst for progesterone 11α-hydroxylation[J].J Biotechnology,1991,60(3):207-216.
[14]Tucker K G,Thomas C R.Inoculum effects on fungal morphology:Shake flask vs.agitated bioreactor[J].Biotechnology Techniques,1994,8(3):153-156.
[15]Shoop W L,Morzik H,Fisher M H.Structure and activity of avermectins and nemadectin in animal health[J].Vet Parasitol,1995,59:139-151.