CN111133098A - 细菌菌体的制备方法 - Google Patents
细菌菌体的制备方法 Download PDFInfo
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- CN111133098A CN111133098A CN201880061970.9A CN201880061970A CN111133098A CN 111133098 A CN111133098 A CN 111133098A CN 201880061970 A CN201880061970 A CN 201880061970A CN 111133098 A CN111133098 A CN 111133098A
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- bacillus
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Abstract
本发明提供细菌菌体的制备方法,所述方法包括:在细菌的多阶段的液体培养工序中,在比最终阶段早的阶段用含有抗生素的液体培养基培养细菌,在最终阶段使抗生素的浓度低于该早的阶段而进行培养的工序。由此可以抑制培养后得到的细菌中形成异常菌落的突变个体的出现,并以良好的生产率制备细菌菌体。
Description
技术领域
本发明涉及对细菌多阶段液体培养期间可能产生的、形成异常菌落的突变个体的产生和增殖进行抑制,并以良好的生产率制备细菌菌体的方法。
背景技术
细菌被利用在酶、有用物质的生产,发酵食品的生产,有机物的分解,肠道调理剂,微生物农药,微生物化肥等各种领域中。
在任一种领域中都需要为了制备菌体而进行细菌的培养,但存在这样的案例:在培养中出现在菌落形态方面显示异常的突变个体,比具有原本性质的个体更优势地增殖,由此在培养结束后的菌体中,原本的有用性质会受损。
例如,在非专利文献1中观察到,在芽孢杆菌(Bacillus)属细菌的液体培养基中的数千代的传代培养中,出现在菌落形态等方面显示异常的突变个体,比原本的个体更优先地增殖,由此会完全将其取代的现象。这样的现象推测为由于因特定的培养环境而发生特异化,剥离不需要的代谢体系的突变个体以比原本的个体更优势地进行增殖而产生。
在非专利文献2中显示:枯草芽孢杆菌(Bacillus subtilis)916株的芽孢菌霉素L(Bacillomycin L)和表面活性素(Surfactin)的生物合成能力缺陷突变株示出与野生型株不同的菌落形态。已知芽孢菌霉素L及表面活性素对一部分的植物疾病显示防治效果,被认为是与菌落形态的突变和有用性质的丧失相关的例子。
另外,在非专利文献3中示出了在用于强化枯草芽孢杆菌G3株的抗菌活性的育种中,在菌落形态与亲本株发生变化的菌株中,Itulin A的产量增加了的例子。
在非专利文献4中显示:对于米氏解硫胺素芽孢杆菌(Aneurinibacillus migulanus)(旧分类为短芽孢杆菌(Bacillus brevis))ATCC9999株,从菌种保藏保管株中检测到6种类型的菌落形态,并且它们的短杆菌肽S(Gramicidin S)生产率不同。
在非专利文献5中显示了枯草芽孢杆菌VT30M株及地衣芽孢杆菌(Bacillus licheniformis)VT3株的菌落形态突变株与野生型相比,显现出不同的酶生产率、抗生素耐受性的例子。
现有技术文献
非专利文献
非专利文献1:Biotechnology progress 2005,21,4,1026-1031
非专利文献2:Appl.Microbiol.Biotechnol.2015,99,41897-1910
非专利文献3:Zhiwu Bingli Xuebao 2008,38,2,185-191
非专利文献4:Appl.Environ.Microbiol.2007,73,20,6620-6628
非专利文献5:Appl.Environ.Microbiol.1989,55,3026-3028
发明内容
发明要解决的问题
如上所述,在细菌的液体培养中,特别是进行多阶段培养的情况中,在培养中出现菌落形态方面显示异常的突变个体,通过比具有原本的性质的个体更优势地增殖,由此培养结束后的菌体中原本的有用性质会受损。尽管存在上述情况,用于抑制这种现象的有效技术至今还没有报告。
因此,本发明的课题在于提供用于在细菌的多阶段的液体培养工序中,对于培养后得到的细菌中在菌落形态方面显示异常的突变个体的出现进行抑制,并以良好的生产率制备细菌菌体的新方法。
解决问题的手段
本发明人为了解决上述课题而进行了深入研究,结果发现,在细菌的多阶段的液体培养工序中,通过在比最终阶段早的阶段中使用包含抗生素的培养基进行培养,在最终阶段使抗生素的浓度低于所述早的阶段而进行培养,由此在培养结束后得到的细菌群体中,菌落形态方面显示异常的突变个体的出现明显降低,并可以以良好的生产率制备细菌菌体,从而完成了本发明。
本发明如下所述。
[1]细菌菌体的制备方法,所述方法包括:在细菌的多阶段的液体培养工序中,在比最终阶段早的阶段用含有抗生素的液体培养基培养细菌,在最终阶段使抗生素的浓度低于该早的阶段而进行培养的工序。
[2]根据[1]所述的方法,其中,细菌为芽孢形成细菌。
[3]根据[2]所述的方法,其中,芽孢形成细菌为芽孢杆菌(Bacillus)属细菌。
[4]根据[3]所述的方法,其中,芽孢形成细菌选自由以下组成的组:枯草芽孢杆菌(Bacillus subtilis)、解淀粉芽孢杆菌(Bacillus amyloliquefaciens)、短小芽孢杆菌(Bacillus pumilus)、简单芽孢杆菌(Bacillus simplex)、迟缓芽孢杆菌(Bacillus lentus)、侧孢芽孢杆菌(Bacilluslaterosporus)、蜂房芽孢杆菌(Bacillus alvei)、日本甲虫芽孢杆菌(Bacilluspopilliae)、地衣芽孢杆菌(Bacillus licheniformis)、短芽孢杆菌(Bacillusbrevis)、嗜热脂肪芽孢杆菌(Bacdlus stearothermophilus)、嗜碱芽孢杆菌(Bacillus alcalophilus)、凝结芽孢杆菌(Bacilluscoagulans)、环状芽孢杆菌(Bacillus circulans)、暹罗芽孢杆菌(Bacillus siamensis)、灿烂芽孢杆菌(Bacillus lautus)、克劳氏芽孢杆菌(Bacillus clausii)、巨大芽孢杆菌(Bacillus megaterium)、苏云金芽孢杆菌(Bacillus thuringiensis)、蜡状芽孢杆菌(Bacillus cereus)、坚强芽孢杆菌(Bacillus firmus)、贝莱斯芽孢杆菌(Bacillus velezensis)、皮基诺伊芽孢杆菌(Bacillus pichinotyi)、酸热芽孢杆菌(Bacillus acidocaldarius)、好碱芽孢杆菌(Bacillus alkalicola)、产氮芽孢杆菌(Bacillusazotoformans)、炭疽芽孢杆菌(Bacillus anthracis)、栗褐芽孢杆菌(Bacillus badius)、巴达维亚芽孢杆菌(Bacillus bataviensis)、环庚基芽孢杆菌(Bacillus cycloheptanicus)、解硫胺素芽孢杆菌(Bacillus aneurinilyticus)、米氏芽孢杆菌(Bacillus migulanus)、深海芽孢杆菌(Bacillus abyssalis)、潮间带芽孢杆菌(Bacillus aestuarii)、多粘芽孢杆菌(Bacillus polymyxa)及芽孢杆菌属(Bacillus sp.)。
[5]根据[1~[4]中任一项所述的方法,其中,抗生素为选自由链霉素、林可霉素、红霉素、利福平、氯霉素、放线菌素、夫西地酸、闰年霉素、嘌呤霉素、壮观霉素、四环素及硫链丝菌素组成的组中的1种以上的抗生素。
[6]根据[5]所述的方法,其中,抗生素为链霉素。
[7]根据[1]~[6]中任一项所述的方法,其中,所述比最终阶段早的阶段的液体培养基中的抗生素的浓度为0.001ppm~10,000ppm。
[8]根据[1]~[6]中任一项所述的方法,其中,所述比最终阶段早的阶段的液体培养基中的抗生素的浓度为1.0~90ppm。
[9]使形成异常菌落的突变个体的出现降低的细菌培养方法,所述方法包括:在细菌的多阶段的液体培养工序中,在比最终阶段早的阶段用含有抗生素的液体培养基培养细菌,在最终阶段使抗生素的浓度低于该早的阶段而进行培养的工序。
[10]在细菌的液体培养期间抑制形成异常菌落的突变个体的出现及增殖的方法,所述方法包括:在细菌的多阶段的液体培养工序中,在比最终阶段早的阶段用含有抗生素的液体培养基培养细菌,在最终阶段使抗生素的浓度低于该早的阶段而进行培养的工序。
发明效果
根据本发明,通过在多阶段培养使用的培养基中含有抗生素并培养细菌,在最终阶段中使抗生素的量降低而培养细菌,可以由此使在细菌的液体培养结束时的菌落形态方面显示异常的突变个体的比率降低,并以良好的生产率制备细菌菌体。由于在菌落形态方面显示异常的突变个体丧失期望性质的可能性高,因此,通过使这样的突变个体降低,可以将通过培养得到的细菌菌体以良好的状态用于酶、有用物质的生产,发酵食品的生产,有机物的分解,肠道调理剂,微生物农药、微生物化肥等用途。
附图说明
[图1]示出异常菌落的形态的一个实例的图(照片)。用○示出异常菌落。
具体实施方式
在本发明中,细菌的种类没有特别的限制,能够液体培养,在固体培养基上形成菌落即可,可以是革兰氏阳性菌,也可以是革兰氏阴性菌,可列举例如:埃希氏菌(Escherichia)属细菌,志贺氏菌(Shigella)属细菌,沙门氏菌(Salmonella)属细菌,克雷伯氏菌(Klebsiella)属细菌,耶尔森氏菌(Yersinia)属细菌,肠杆菌(Enterobacter)属细菌,假单胞菌(Pseudomonas)属细菌,布鲁氏菌(Brucella)属细菌,葡萄球菌(Staphylococcus)属细菌,链球菌(Streptococcus)属细菌,链霉菌(Streptomyces)属细菌,红球菌(Rhodococcus)属细菌,醋酸杆菌(Acetobacterium)属细菌,甲烷杆菌(Methanobacterium)属细菌,肠球菌(Enterococcus)属细菌,芽孢杆菌(Bacillus)属细菌,梭菌(Closttridium)属细菌,棒状杆菌(Corynebacterium)属细菌,分枝杆菌(Mycobacterium)属细菌等。
这些细菌中,芽孢杆菌(Bacillus)属细菌,类芽孢杆菌(Paenibacillus)属细菌,土芽孢杆菌(Geobacillus)属细菌,梭菌(Clostridium)属,芽孢八叠球菌(Sporosarcina)属等芽孢形成细菌是更优选的。
作为芽孢杆菌属细菌没有特别限制,只要是分类为芽孢杆菌属的细菌即可,可列举例如:枯草芽孢杆菌(Bacillus subtilis),解淀粉芽孢杆菌(Bacillus amyloliquefaciens),短小芽孢杆菌(Bacillus pumilus),简单芽孢杆菌(Bacillus simplex),迟缓芽孢杆菌(Bacillus lentus),侧孢芽孢杆菌(Bacillus laterosporus),蜂房芽孢杆菌(Bacillus alvei),日本甲虫芽孢杆菌(Bacillus popilliae),地衣芽孢杆菌(Bacillus licheniformis),短芽孢杆菌(Bacillus brevis),嗜热脂肪芽孢杆菌(Bacillus stearothermophilus),嗜碱芽孢杆菌(Bacillus alcalophilus),凝结芽孢杆菌(Bacillus coagulans),环状芽孢杆菌(Bacillus circulans),暹罗芽孢杆菌(Bacillus siamensis),灿烂芽孢杆菌(Bacillus lautus),克劳氏芽孢杆菌(Bacillus clausii),巨大芽孢杆菌(Bacillus megaterium),苏云金芽孢杆菌(Bacillus thuringiensis),蜡状芽孢杆菌(Bacillus cereus),坚强芽孢杆菌(Bacdlus firmus),贝莱斯芽孢杆菌(Bacillus velezensis),皮基诺伊芽孢杆菌(Bacillus pichinotyi),酸热芽孢杆菌(Bacillus acidocaldarius),好碱芽孢杆菌(Bacillus alkalicola),产氮芽孢杆菌(Bacillus azotoformans),炭疽芽孢杆菌(Bacillus anthracis),栗褐芽孢杆菌(Bacillus badius),巴达维亚芽孢杆菌(Bacillus bataviensis),环庚基芽孢杆菌(Bacillus cycloheptanicus),解硫胺素芽孢杆菌(Bacillus aneurinilyticus),米氏芽孢杆菌(Bacillus migulanus),深海芽孢杆菌(Bacillus abyssalis),潮间带芽孢杆菌(Bacillus aestuarii),多粘芽孢杆菌(Bacillus polymyxa),或芽孢杆菌属(Bacillus sp.)。
作为类芽孢杆菌属细菌,可列举:浸麻类芽孢杆菌(Paenibacillus macerans),解淀粉类芽孢杆菌(Paenibacillus amylolyticus),皮尔瑞俄类芽孢杆菌(Paenibacillus peoriate),埃吉类芽孢杆菌(Paenibacillus elgii)等。
作为土芽孢杆菌属细菌,可列举热葡糖苷酶土芽孢杆菌(Geobacillus thermoglucosidasius),热解木糖土芽孢杆菌(Geobacillus caldoxylosilyticus),嗜热脂肪土芽孢杆菌(Geobacillus stearothermophilus)等。
作为梭菌属细菌,可列举:丁酸梭菌(Closttridium butyricum),、克鲁维氏梭菌(Clostridium kluyveri),丙酮丁醇梭菌(Closttridium acetobutylicum),氨基丁酸梭菌(Closttridium aminobutyricum),拜氏梭菌(Closttridium beijerinckii),糖多丁醇乙酸梭菌(Closttridium saccharoperbutylacetonicum),、热纤梭菌(Closttridium thermocellum),杨氏梭菌(Closttridium ljungdahlii),肉毒梭菌(Closttridium botulinum)等。
作为芽孢八叠球菌属细菌,可列举:巴氏芽孢八叠球菌(Sporosarcina pasteurii),脲芽孢八叠球菌(Sporosarcina ureae),嗜冷芽孢八叠球菌(Sporosarcina psychrophila),耐热芽孢八叠球菌(Sporosarcina thermotolerans)等。
在本发明的方法中培养的细菌可以为非基因重组细菌,也可以为基因重组细菌,但优选为不包含抗生素耐受性基因的细菌。
另外,在本发明的方法中培养的细菌可以为野生型也可以为突变型,即使在为后者的情况下,也优选不具有抗生素耐受性突变的细菌。
在本发明的细菌菌体的制备方法及细菌的培养方法中,包括将细菌在含有抗生素的液体培养基中培养的工序。
液体培养基可以根据培养对象的细菌的种类适当选择,可以使用适于细菌的培养的浓度的包含碳源及氮源等培养基成分的一般液体培养基。
例如,作为碳源,可例示糖(淀粉,葡萄糖,乳糖,甘油,阿拉伯糖,核糖,木糖,半乳糖,果糖,甘露糖,肌醇,甘露醇,山梨糖醇,葡糖胺,N-乙酰葡糖胺,纤维二糖,麦芽糖,蔗糖,海藻糖,木糖醇等)或者糖源原料,醇,有机酸,有机酸盐,烷烃或其它一般的碳源,作为氮源,可例示源自大豆的成分,源自酵母的成分,源自玉米的成分,动植物蛋白质及其分解物,硝酸铵,硫酸铵,氯化铵,乙酸铵等铵盐,氨,硝酸钠,硝酸钾,谷氨酸钠,尿素等。
作为碳源及氮源以外的培养基成分,可以举出微量金属盐,氨基酸,维生素等,可以根据需要适当添加。
在本发明的方法中,作为抗生素没有特别限定,只要能够将异常菌落出现率比非添加时降低即可,具体而言可例示:链霉素,林可霉素,红霉素,利福平,氯霉素,放线菌素,夫西地酸,闰年霉素,嘌呤霉素,壮观霉素,四环素,硫链丝菌素。这其中特别优选链霉素。
抗生素为优选为不妨碍细菌的生长(亚致死量以下)的浓度,优选以使得液体培养基中抗生素的浓度为0.001ppm以上10,000ppm以下的方式添加。
抗生素的浓度优选为对于细菌的增殖阻碍浓度以下,也根据细菌的种类而异,例如优选为1,000ppm以下,更优选300ppm以下,进一步优选90ppm以下。另一方面,抗生素的浓度的下限为能抑制异常菌落的出现的浓度即可,优选为0.01ppm以上,更优选为0.1ppm以上,进一步优选为1ppm以上。
抗生素在比最终阶段早的阶段添加到液体培养基中。抗生素可以从培养开始时含在液体培养基中,也可以在培养途中添加。在培养途中添加时,优选在菌体以一定程度增殖的阶段添加,例如优选在从培养开始1~10小时之间添加。另外,液体培养基中抗生素的浓度也可以在培养途中进行补充等而变化。
液体培养工序包括伴随继代培养的2个阶段以上的培养。只要是2个阶段以上即可,培养阶段数为几个阶段都可以,优选可以为2~4个阶段的多阶段培养。
需要说明的是,也可以是第1阶段为1个培养容器,第2阶段为4个培养容器这样的方式,随着进入后面阶段而增加培养容器的数量。另外,也可以是第1阶段为1L,第2阶段为10L这样的方式,随着进入后段而增加培养容器的容量。
对于多阶段培养的培养时间,例如在进行2个阶段的培养的情况下,可以举出将细菌接种在第1阶段的培养基中进行10~40小时、优选为20~30小时的培养,将在第1阶段的培养中得到的菌体的一部分接种在第2阶段的培养基中,进行15~80小时、优选为20~50小时的培养的实施方式,作为总的培养时间,优选为25~120小时,更优选为30~80小时。
例如,在进行3个阶段的培养的情况下,可以举出将细菌接种在第1阶段的培养基中进行10~40小时、优选为20~30小时的培养,将第1阶段的培养中得到的菌体的一部分接种在第2阶段的培养基中,进行5~40小时、优选为10~30小时的培养,将第2阶段的培养中得到的菌体的一部分接种在第3阶段的培养基中,进行15~80小时、优选为20~50小时的培养的实施方式,作为总的培养时间,优选为30~160小时,更优选为35~110小时。
例如在进行4个阶段的培养的情况下,可以举出将细菌接种在第1阶段的培养基中进行10~40小时、优选为20~30小时的培养,将第1阶段的培养中得到的菌体的一部分接种在第2阶段的培养基中,进行5~40小时、优选为10~30小时的培养,将第2阶段的培养中得到的菌体的一部分接种在第3阶段的培养基中,进行5~40小时、优选为10~30小时的培养,将第3阶段的培养中得到的菌体的一部分接种在第4阶段的培养基中,进行15~80小时、优选为20~50小时的培养的实施方式,作为总的培养时间,优选为35~200小时,更优选为60~140小时。
像这样,在以多阶段进行液体培养的情况下,只要其中的除去最终阶段以外的至少任意1个阶段中使用含有抗生素的培养基进行培养,在最终阶段中以低于在含有抗生素的阶段中的抗生素浓度进行培养即可。通过在最终阶段将抗生素浓度降低,使得将引起阻碍增殖、阻碍芽孢形成的因素消除,因此,可以更迅速地推进培养工序。通常在利用微生物的多阶段培养进行的生产中,一般而言,最终阶段是为规模最大的,因此,在最终阶段中降低抗生素浓度,也关系到降低制造成本。
例如,关于最终阶段中的抗生素的浓度,可以使其为含有抗生素的阶段中的抗生素的浓度的1/2以下,1/5以下,1/10以下,1/50以下,或1/100以下,也可以使最终阶段中的抗生素的浓度为零(检测限以下)。
例如,也可以为后述的实施例1那样,将培养以3个阶段进行,在第1阶段和第2阶段添加抗生素,在第3阶段不添加抗生素,将第2阶段的培养液进行1%继代培养,由此使得抗生素浓度降低至第2阶段的百分之一的培养方法。
另外,也可以将抗生素的浓度按每个阶段逐渐减少,在该情况下,最终阶段中的抗生素的浓度可以为最大浓度的1/2以下,1/5以下,1/10以下,1/50以下,或1/100以下。
需要说明的是,对使用含有抗生素的培养基培养的时期而言,优选为总计10小时以上。
培养温度可以根据细菌的种类适宜选择,例如为10℃~50℃、优选为15℃~50℃,更优选为15℃~40℃。
对于其它的氧浓度,pH等各种条件而言,只要为常规的用于细菌的液体培养的条件即可,可例示例如:于有氧条件(例如:氧浓度15~50%)中,同时进行搅拌的培养条件。培养基的pH优选为6.5~8.5,更优选为7.0~8.0。
对于是否有异常菌落出现,可以通过在使用抗生素的液体培养后,提取得到的菌体的一部分,将其稀释为菌落彼此不粘接、能识别菌落形态的程度的浓度,涂布在琼脂培养基等固体培养基上培养,并观察出现的菌落的形态,由此确认。
异常菌落是指显示与野生型的细菌的菌落形态为不同形态的菌落。可列举例如,如表3所示,正常型菌落的形态为大致圆形,侧面为透镜样,并且表面为平滑或粘液样,与此相对,异常菌落为诸如形态不规则、侧面扁平,表面为粗糙面的情况。符合形态面的异常,侧面的异常,表面的异常中的1个以上,则可以称为异常菌落。图1示出异常菌落的形态的一个实例,以圆所圈的那些为异常菌落。
实施例
以下结合实施例对本发明进行具体说明,但本发明不受限于以下实施例。
(实施例1)
使用500mL锥形瓶(带挡板),分别制成各自为100mL的包含表1中记载的培养基成分的液体培养基,进行高压釜灭菌。需要说明的是,为了避免美拉德(Maillard)反应,葡萄糖在另外灭菌的基础上,进行无菌混合。
表1培养基组成
成分 | 制造商 | 浓度(g/L) |
葡萄糖 | 和光纯药 | 25.0 |
脱脂大豆粉 | AJINOMOTO HEALTHY SUPPLY | 20.0 |
玉米浸渍液 | Difco | 5.0 |
酵母提取物 | Roquette | 4.0 |
MnCl<sub>2</sub>·4H<sub>2</sub>O | Difco | 0.18 |
NaCl | 和光纯药 | 1.00 |
KH<sub>2</sub>PO<sub>4</sub> | 和光纯药 | 0.50 |
MgSO<sub>4</sub>·7H<sub>2</sub>O | 和光纯药 | 0.63 |
CaCl<sub>2</sub> | 和光纯药 | 0.19 |
FeSO<sub>4</sub> | 和光纯药 | 0.00038 |
按照表2中记载的那样设定试验区。依照各试验区的条件,将过滤器灭菌后的链霉素水溶液无菌地添加到培养基中。
从普通琼脂培养基上生长的枯草芽孢杆菌ITB105株(NITE BP-01727)的菌落取1白金环进行接种之后,于30℃,进行150rpm振荡培养(第1阶段)。18小时后,分别分取1mL继代培养到新的培养基中,同样地进行了振荡培养(第2阶段)。再过24小时后,分别分取1mL继代培养到新的培养基中,同样地进行了振荡培养(第3阶段)。对于第3阶段的培养液,在30小时后进行取样。
麦2试验区的设定
对于得到的培养液,用灭菌水稀释到1×107倍,涂布100μL在普通琼脂培养基上,于37℃过夜培养。测量菌落的出现数,算出CFU。另外,对于出现的菌落,依照表3的标准将与亲本株的菌落形态显然不同的那些作为异常菌落而进行出现数的测量,算出占全部的比率。将其结果示于表4。其结果,在3个阶段培养中,在完全未加入抗生素的情况下,异常菌落出现了约5%,在第1阶段及第2阶段加入链霉素,第3阶段未加入的培养情况下,没有观察到异常菌落的出现。另外,在第1阶段,第2阶段,第3阶段的全部中加入链霉素的情况下,虽然没有出现异常菌落,但菌落的出现数及正常型菌落的出现数低,与试验区编号2~6相比,细菌菌体的生产率降低了。
表3菌落形态的判定基准
表4试验结果
Claims (10)
1.细菌菌体的制造方法,所述方法包括:在细菌的多阶段的液体培养工序中,在比最终阶段早的阶段用含有抗生素的液体培养基培养细菌,在最终阶段使抗生素的浓度低于该早的阶段而进行培养的工序。
2.根据权利要求1所述的方法,其中,细菌为芽孢形成细菌。
3.根据权利要求2所述的方法,其中,芽孢形成细菌为芽孢杆菌(Bacillus)属细菌。
4.根据权利要求3所述的方法,其中,芽孢形成细菌选自由以下组成的组:枯草芽孢杆菌(Bacillus subtilis)、解淀粉芽孢杆菌(Bacillus amyloliquefaciens)、短小芽孢杆菌(Bacillus pumilus)、简单芽孢杆菌(Bacillus simplex)、迟缓芽孢杆菌(Bacilluslentus)、侧孢芽孢杆菌(Bacillus laterosporus)、蜂房芽孢杆菌(Bacillus alvei)、日本甲虫芽孢杆菌(Bacillus popilliae)、地衣芽孢杆菌(Bacillus licheniformis)、短芽孢杆菌(Bacillus brevis)、嗜热脂肪芽孢杆菌(Bacdlus stearothermophilus)、嗜碱芽孢杆菌(Bacillus alcalophilus)、凝结芽孢杆菌(Bacillus coagulans)、环状芽孢杆菌(Bacillus circulans)、暹罗芽孢杆菌(Bacillus siamensis)、灿烂芽孢杆菌(Bacilluslautus)、克劳氏芽孢杆菌(Bacillus clausii)、巨大芽孢杆菌(Bacillus megaterium)、苏云金芽孢杆菌(Bacillus thuringiensis)、蜡状芽孢杆菌(Bacillus cereus)、坚强芽孢杆菌(Bacillus firmus)、贝莱斯芽孢杆菌(Bacillus velezensis)、皮基诺伊芽孢杆菌(Bacillus pichinotyi)、酸热芽孢杆菌(Bacillus acidocaldarius)、好碱芽孢杆菌(Bacillus alkalicola)、产氮芽孢杆菌(Bacillus azotoformans)、炭疽芽孢杆菌(Bacillus anthracis)、栗褐芽孢杆菌(Bacillus badius)、巴达维亚芽孢杆菌(Bacillusbataviensis)、环庚基芽孢杆菌(Bacillus cycloheptanicus)、解硫胺素芽孢杆菌(Bacillus aneurinilyticus)、米氏芽孢杆菌(Bacillus migulanus)、深海芽孢杆菌(Bacillus abyssalis)、潮间带芽孢杆菌(Bacillus aestuarii)、多粘芽孢杆菌(Bacilluspolymyxa)及芽孢杆菌属(Bacillus sp.)。
5.根据权利要求1~4中任一项所述的方法,其中,抗生素为选自由链霉素、林可霉素、红霉素、利福平、氯霉素、放线菌素、夫西地酸、闰年霉素、嘌呤霉素、壮观霉素、四环素及硫链丝菌素组成的组中的1种以上的抗生素。
6.根据权利要求5所述的方法,其中,抗生素为链霉素。
7.根据权利要求1~6中任一项所述的方法,其中,所述比最终阶段早的阶段的液体培养基中的抗生素的浓度为0.001ppm~10,000ppm。
8.根据权利要求1~6中任一项所述的方法,其中,所述比最终阶段早的阶段的液体培养基中的抗生素的浓度为1.0~90ppm。
9.使形成异常菌落的突变个体的出现降低的细菌培养方法,所述方法包括:在细菌的多阶段的液体培养工序中,在比最终阶段早的阶段用含有抗生素的液体培养基培养细菌,在最终阶段使抗生素的浓度低于该早的阶段而进行培养的工序。
10.在细菌的液体培养期间抑制形成异常菌落的突变个体的出现及增殖的方法,所述方法包括:在细菌的多阶段的液体培养工序中,在比最终阶段早的阶段用含有抗生素的液体培养基培养细菌,在最终阶段使抗生素的浓度低于该早的阶段而进行培养的工序。
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