CN108203697B - 一种鱼类天然杀伤细胞增强因子的酵母工程菌及其应用 - Google Patents
一种鱼类天然杀伤细胞增强因子的酵母工程菌及其应用 Download PDFInfo
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Abstract
本发明公开了一种鱼类天然杀伤细胞增强因子的酵母工程菌及应用,产河豚鱼NKEF酵母工程菌命名为酿酒酵母菌INVSC1‑pYES2‑NKEF,保藏号:CGMCC1847。该工程菌是通过将河豚鱼NKEF基因插入pYES2载体的EcoRI和XhoI酶切位点之间,构建了重组质粒,再将重组质粒导入表达宿主酿酒酵母中得到的;将所述酵母工程菌在30℃条件下经2%半乳糖诱导培养,NKEF重组蛋白的表达量可达20%以上。本发明还公开了上述酵母工程菌在水产动物养殖中的抗病应用,可实现提高水产养殖动物抗感染能力和自身免疫能力的作用。
Description
技术领域
本发明涉及水产生物技术和水产养殖病害防治领域,具体是一种鱼类天然杀伤细胞增强因子的酵母工程菌及其应用。
背景技术
随着水产养殖业的高速发展,疫病已成为水产养殖业发展的重大制约因素之一。
长期滥用药物不仅使药物的使用效果受到限制,延误治病,造成大量死亡和药物投入的双重损失;还可诱发病菌基因突变或转移而产生抗药性,导致无药可用;更严重的是药物在养殖动物体内残留超标的教训极为惨重,养殖业赖于持续发展的某些生态资源枯竭化。尤其在我国加入WTO以后,对环境和食品卫生安全的要求和标准越来越高,一大批抗生素已被明令禁用,水产动物病害防治已面临无药可用的尴尬境地。因此,研制开发绿色、安全、高效的水产动物病害防治药物替代危害性大的化学药品已势在必行。
发明内容
本发明的目的之一在于提供一种高效表达河豚鱼NKEF重组蛋白的鱼类天然杀伤细胞增强因子的酵母工程菌。
本发明的目的之二在于提供一种鱼类天然杀伤细胞增强因子的酵母工程菌的应用,用于水产动物养殖中抗病的用途,能明显提高养殖水产动物的成活率和自身免疫能力,降低养殖成本,大大减少病害防治中抗生素和药物的使用量,保障养殖动物食品安全和人类健康。
本发明针对背景技术中提到的问题,采取的技术方案为:
酿酒酵母 INVSC1购于上海北诺生物科技有限公司。酵母工程菌INVSC1-pYES2-NKEF的保藏号为CGMCC1847。
酵母工程菌的构建方法包括以下步骤:
1)根据GeneBank数据库中登记的青斑河豚鱼NKEF基因序列Genbank No.DQ003333.1,设计表达载体构建引物P1和P2:
P1:5'-GGGAATTCAAAATGGCTGCAGGC-3' (-sense含EcoRI)
P2:5'-CCGCTCGAGTTAGTGCTTGGAGAAG-3' (-sense含XhoI)
2)使用Trizol试剂盒提取河豚鱼脾脏总RNA,使用逆转录试剂盒cDNA SynthesisKit (M-MLV Version)将总RNA逆转录成cDNA模版;
3)以步骤2)得到的河豚鱼cDNA序列为模板,通过PCR扩增获得NKEF基因片段;
PCR体系:10×PCR Buffer 2μl,dNTP Mixture 0.4μl,Primer1和2各 0.8μl,cDNA模版0.2μl,Ex Taq polymerase 0.2μl,加ddH2O补足至20μl;
PCR参数:94℃预变性4min,94℃变性30s,58℃退火30s,72℃延伸30s,32个循环,72℃后延伸10min,15℃ 10min;
4)用限制性内切酶EcoRI和XhoI双酶切NKEF基因片段,纯化回收后与同样经双酶切处理的pYES2载体连接,转化大肠杆菌TOP10,获得到重组质粒pYES2-NKEF,选取阳性克隆;
5)将鉴定后的阳性克隆质粒电转化导入酿酒酵母 INVSC1菌株中,获得酵母工程菌INVSC1-pYES2-NKEF。上述酵母工程菌INVSC1-pYES2-NKEF含有河豚鱼NKEF基因的重组质粒,在30℃条件下,经2%半乳糖诱导后可见在分子量28kDa的位置有明显的NKEF重组蛋白条带,经薄层扫描分析,NKEF重组蛋白的表达量可达20%以上。
一种鱼类天然杀伤细胞增强因子的酵母工程菌的应用,用于水产动物养殖中抗病的用途。NKEF-A和NKEF-B分别属于PrxⅠ和PrxⅡ抗氧化蛋白亚家族,其氨基酸序列的N端和C端都含有在进化上高度保守的半胱氨酸,可清除细胞在应激状况下产生的多余的活性氧,保护体内重要的蛋白质、脂类及DNA等分子免遭氧化剂的损伤。能够介导细胞对促炎分子的应答,参与机体的抗感染免疫,能够抑制脂多糖和低密度脂蛋白诱导的炎症反应。但只有还原型的NKEF-A能增强NK细胞对肿瘤细胞的细胞毒活性。
将所述酵母工程菌INVSC1-pYES2-NKEF活化后接种至YPD培养基和发酵培养基中进行一级种和二级种摇瓶培养,取二级种培养物接种至50L全自动发酵罐中进行分批补料培养,最大程度激活其NKEF基因片段的表达,待所述酵母工程菌培养结束后收集菌体,用蒸馏水清洗后进行超声破碎处理,气流干燥制成酵母菌粉用于水产动物养殖中抗病的用途。上述酵母菌粉能显著提高养殖水产动物的成活率和自身免疫能力,降低养殖成本,大大减少病害防治中抗生素和药物的使用量,保障养殖动物食品安全和人类健康,具有重大的社会经济效益。
为优化上述技术方案,采取的措施还包括:发酵培养基成分及其重量份为:蔗糖90~120份、酵母粉20~35份、蛋白胨4~6份、尿素10~20份、麦芽汁2~4份、黄烯醇0.001~0.003份、KH2PO4 2~5份、K2HPO4·3H2O 2.0~3.5份、MgSO4·7H2O 0.06~0.2份、ZnSO4·7H2O 0.001~0.004份、MnSO4·4H2O 0.008~0.02份、木糖醇0.001~0.003份、生物素0.002~0.006份。上述发酵培养基不仅能充分提供酵母工程菌INVSC1-pYES2-NKEF发酵所需的碳源、氮源及各种生长因子,还能提高酵母工程菌INVSC1-pYES2-NKEF的NKEF基因的mmRNA和amRNA表达量,提高还原型NKEF-A的产量。还原型NKEF-A可通过激活NK细胞和靶细胞膜表面的某些结构,促进两者结合,从而增强 NK 细胞杀伤活力。上述发酵培养基中各成分有相互协同作用,发酵结束后酵母工程菌INVSC1-pYES2-NKEF菌体中NKEF重组蛋白的表达量可达35.7%以上,并且还原型NKEF-A的含量高。
作为优选,一级种摇瓶培养条件为:培养温度25~35℃、摇床转速100~300r/min、培养时间18~24h;二级种摇瓶培养条件为:接种量8~12%(V/V)、培养温度25~35℃、摇床转速100~300r/min、培养时间20~28h。50L全自动发酵罐培养条件为:发酵培养基初始OD值为0.4~0.6,并补加1.4~2.5%半乳糖,溶氧在30%以上,pH4.5~5.5,培养温度25~35℃,发酵时间36~72h;所述分批补料方法为:发酵10~15h后第一次补加培养基,并添加15~25%甘油,14~18h后第二次补加培养基,22~26h后第三次补加培养基,至发酵结束。
作为优选,超声破碎条件为:超声破碎频率为15~25kHz,破碎时间为17~23min,时间间隔为10:8s/s,气流干燥温度为25~30℃。
与现有技术相比,本发明的优点在于:本发明将青斑河豚鱼NKEF基因定向克隆到E.coli—酵母穿梭表达质粒pYES2中,经电转化入酿酒酵母Saccharomyces cerevisiae,获得一株高效表达河豚鱼NKEF重组蛋白的酵母工程菌INVSC1-pYES2-NKEF。酵母工程菌INVSC1-pYES2-NKEF采用本发明发酵培养基发酵培养后,NKEF重组蛋白的表达量可达35.7%,并且还原型NKEF-A的含量高。本发明制备的酵母菌粉能显著提高养殖水产动物的成活率和自身免疫能力,降低养殖成本,大大减少病害防治中抗生素和药物的使用量,保障养殖动物食品安全和人类健康,具有重大的社会经济效益。
附图说明
图1是重组质粒pYES2-NKEF的构建图;
图2是酵母重组子菌落PCR鉴定图;
图3是重组质粒pYES2-NKEF双酶切鉴定图;
图4是NKEF重组蛋白诱导表达电泳图;
图5是NKEF重组蛋白分离纯化电泳图。
附图标记说明:图4中第1-7列分别为诱导0、4、6、12、18、24、30小时的NKEF表达情况,箭头所指为目的蛋白;图5中第1列为Marker,第1列为蛋白提取物;第2-4列为逐级分离,第5-7列为纯化蛋白。
具体实施方式
下面通过附图和实施例对本发明方案作进一步说明:
实施例1:
(1)河豚鱼NKEF基因的获取
取青斑河豚鱼脾脏组织100mg左右,在液氮中将组织研磨成粉末,用Invitrogen公司的Trizol试剂盒提取总RNA,提取方法按说明书进行。然后用TaKaRa公司逆转录试剂盒cDNA Synthesis Kit (M-MLV Version)将总RNA逆转录成cDNA模版;
根据青斑河豚鱼NKEF基因序列(Genbank No. DQ003333.1),设计表达载体构建引物P1和P2,上游自起始密码子ATG前开始引入EcoRI酶切位点,下游自终止密码子TAA后开始引入XhoI酶切位点;
P1:5'-GGGAATTCAAAATGGCTCAGGC-3'(-sense含EcoRI)
P2:5'-CCGCTCGAGTTAGTGCTTGGAGAAG-3'(-sense含XhoI)
取逆转录产物(cDNA)为模版,利用20μl体系进行PCR扩增,PCR产物回收纯化后得到NKEF基因;PCR体系:10×PCR Buffer 2μl,2.5mmol/L dNTP 0.4μl,引物P1和P2各 0.8μl,cDNA模版0.2μl,Ex Taq polymerase 0.2μl,加ddH2O补足至20μl;PCR参数:94℃预变性4min,94℃变性30s,58℃退火30s,72℃延伸30s,32个循环,72℃后延伸10min,15℃ 10min;PCR产物回收纯化使用胶回收试剂盒(购自Omega公司);
(2)重组质粒的构建
用限制性内切酶EcoRI和XhoI双酶切NKEF基因片段,纯化回收后重组入带有酵母GAL1启动子的E.coli—酵母穿梭表达质粒pYES2,转化大肠杆菌Top10。菌落PCR检测筛选阳性菌落,然后用碱裂解法提取重组表达质粒,进行测序鉴定,结果证明得到插入方向正确的重组表达质粒,含完整的NKEF基因,长度约617bp,基因5'端有EcoRI酶切位点,3'端有XhoI酶切位点。将获得的重组质粒命名为pYES2-NKEF。重组质粒的构建如附图1所示;
菌落PCR检测所用引物为pYES2-F和pYES2-R(根据pYES2通用序列设计),反应体系:Taq DNA聚合酶0.5U,10×PCR Buffer 1μl,2.5mmol/L dNTP 0.4μl,引物pYES2-F和pYES2-R各 1μmol/L,加ddH2O 至10μl。反应条件:94℃ 4min;94℃ 30s,53℃ 30s,72℃1min,35个循环;72℃ 10min;
pYES2-F:5'-AAAACCCCGGATCGGACTAC-3'
pYES2-R:5'-GGGAGGGCGTGAATGTAAGC-3'
(3)重组质粒的转化
碱裂解法提取少量重组质粒pYES2-NKEF,同时制备酿酒酵母感受态细胞,用电脉冲转化方法将重组质粒转入酿酒酵母,方法如下:将40μl酵母悬浮液与5μl质粒DNA混合于预冷的电转化杯(0.2cm),轻摇后冰浴5min;脉冲参数:V=1.5kV,25μF,200 Ohms,4-5ms;电转化后立刻加1ml预冷的1mol/L山梨醇,涂布SC-U筛选平板上(200μl每平板),30℃培养,直至单个菌落出现;
(4)酵母重组子的筛选和鉴定
获得的酵母重组子经菌落PCR检测,筛选长度正确的阳性克隆子。然后提取酵母质粒DNA,重新转化大肠杆菌Top10,扩增质粒后用EcoRI和XhoI双酶切检验,结果如附图2和3所示。将获得的酵母工程菌命名为INVSC1-pYES2-NKEF;
(5)重组NKEF在酿酒酵母中的表达
挑取单菌落酵母重组子,接种至含2%半乳糖的15ml SC-U培养基中,30℃振荡培养过夜,测OD600值。取一定量过夜培养物,4℃、1500g离心5min,收集细胞,用1~2ml诱导培养基重悬细胞,接种于50ml诱导培养基,初始OD值为0.4,30℃振荡培养。在0、4、6、12、18、24、30h收集细胞,检测重组蛋白的表达情况。结果如附图4所示;
重组蛋白提取使用裂解液悬浮酵母细胞,将OD600值调整到50~100,加入500μl裂解液和等体积玻璃珠(Sigma G-8772),振荡30s,冰浴30s,重复4次裂解细胞,然后取部分镜检,观察细胞破碎效果,高速离心10min,收集上清液,用SDS-PAGE电泳分离,薄层色谱扫描仪测定重组NKEF的相对表达量(%);
结果显示,经2%半乳糖诱导后可见在分子量28kDa的位置有明显的NKEF重组蛋白条带,而转化空白质粒的对照菌株没有出现相应的蛋白条带。经薄层扫描分析,NKEF重组蛋白的表达量可达20%以上。结果如附图5所示;
(6)发酵生产NKEF重组蛋白
将保存的INVSC1-pYES2-NKEF工程菌经SC-U斜面培养基,30℃培养活化后,接种至装有10ml种子培养基(YPD)的250ml三角瓶中进行一级种摇瓶培养,培养条件:温度:30℃、摇床转速:200r/min、培养时间:20h;取一级种培养物按10%(V/V)的接种量,接种至装有50ml发酵培养基的250ml三角瓶中进行二级种摇瓶培养,培养条件:温度:30℃、摇床转速:200r/min、培养时间:24h;取二级种培养物按发酵罐工作体积的10%,接种至50L全自动控制发酵罐中,发酵培养基初始OD值需达到0.5,补加2%半乳糖,调节转速和空气流量控制溶氧在30%以上,发酵过程添加NaOH控制pH5.0左右,30℃培养。期间分批补充培养基,12h后第一次补加培养基,并添加20%甘油,16h后第二次补加培养基,24h后第三次补加培养基,发酵至48h。发酵结束后采用离心法(5000rpm,20min)收集酵母细胞,15~25kHz超声破碎,25~30℃气流干燥,制成酵母菌粉;
取适量菌粉,加入SDS-PAGE样品缓冲液,用SDS-PAGE电泳分离,薄层色谱扫描仪测定重组NKEF的相对表达量(%);
(7)含NKEF重组蛋白饲料添加剂的研制
按每公斤鱼(虾)人工配合饲料含10mgNKEF重组蛋白的剂量,添加含NKEF的酵母工程菌粉,以NKEF重组蛋白在酵母中的表达量达20%计,在每公斤鱼(虾)人工配合饲料中添加50mg酵母菌粉;
(8)NKEF重组蛋白在鲫鱼养殖中的应用
试验在6~9月份进行,将养殖鲫鱼网箱分成8组,5组网箱为试验组,3组为对照组,使用含NKEF的饲料(每公斤饲料添加含NKEF工程菌粉50mg)投喂,连续投喂8周,统计自然发病死亡率,计算相对免疫保护率(Relative Percent Survival, RPS),RPS=(1-免疫组死亡率/对照组死亡率)×100%。结果显示,实验组鲫鱼的病害发生率显著下降,平均成活率提高21.20%,相对免疫保护率RPS达70.90%,表明饲喂NKEF重组蛋白能增强鲫鱼在养殖中对病菌感染的抵抗力(表1);
表1 鲫鱼养殖抗病试验结果
(9)NKEF重组蛋白在鲈鱼养殖中的应用
试验在6~9月份进行,将养殖鲈鱼网箱分成8组,5组网箱为试验组,3组为对照组,使用含NKEF的饲料(每公斤饲料添加含NKEF工程菌粉50mg)投喂,连续投喂8周,统计自然发病死亡率,计算相对免疫保护率RPS。结果显示,实验组鲈鱼的发病率显著下降,平均成活率提高25.23%,相对免疫保护率RPS达67.41%,表明以饲喂NKEF重组蛋白能增强鲈鱼对病原菌感染的抵抗力(表2)。
表2 鲈鱼养殖抗病试验结果
实施例2:
酵母工程菌的构建方法包括以下步骤:
1)根据GeneBank数据库中登记的青斑河豚鱼NKEF基因序列Genbank No.DQ003333.1,设计表达载体构建引物P1和P2:
P1:5'-GGGAATTCAAAATGGCTGCAGGC-3' (-sense含EcoRI)
P2:5'-CCGCTCGAGTTAGTGCTTGGAGAAG-3' (-sense含XhoI)
2)使用Trizol试剂盒提取河豚鱼脾脏总RNA,使用逆转录试剂盒cDNA SynthesisKit (M-MLV Version)将总RNA逆转录成cDNA模版;
3)以步骤2)得到的河豚鱼cDNA序列为模板,通过PCR扩增获得NKEF基因片段;
PCR体系:10×PCR Buffer 2μl,dNTP Mixture 0.4μl,Primer1和2各 0.8μl,cDNA模版0.2μl,Ex Taq polymerase 0.2μl,加ddH2O补足至20μl;
PCR参数:94℃预变性4min,94℃变性30s,58℃退火30s,72℃延伸30s,32个循环,72℃后延伸10min,15℃ 10min;
4)用限制性内切酶EcoRI和XhoI双酶切NKEF基因片段,纯化回收后与同样经双酶切处理的pYES2载体连接,转化大肠杆菌TOP10,获得到重组质粒pYES2-NKEF,选取阳性克隆;
5)将鉴定后的阳性克隆质粒电转化导入酿酒酵母 INVSC1菌株中,获得酵母工程菌INVSC1-pYES2-NKEF;
6)将所述酵母工程菌INVSC1-pYES2-NKEF活化后接种至YPD培养基和发酵培养基中进行一级种和二级种摇瓶培养,取二级种培养物接种至50L全自动发酵罐中进行分批补料培养,最大程度激活其NKEF基因片段的表达,待所述酵母工程菌培养结束后收集菌体,用蒸馏水清洗后进行超声破碎处理,气流干燥制成酵母菌粉用于水产动物养殖中抗病的用途。发酵培养基成分及其重量份为:蔗糖100份、酵母粉30份、蛋白胨5份、尿素15份、麦芽汁3份、黄烯醇0.002份、KH2PO44份、K2HPO4·3H2O 2.6份、MgSO4·7H2O 0.1份、ZnSO4·7H2O0.0025份、MnSO4·4H2O 0.0125份、木糖醇0.001份、生物素0.004份。一级种摇瓶培养条件为:培养温度30℃、摇床转速200r/min、培养时间20h;二级种摇瓶培养条件为:接种量10%(V/V)、培养温度30℃、摇床转速200r/min、培养时间24h。50L全自动发酵罐培养条件为:发酵培养基初始OD值为0.5,并补加2%半乳糖,溶氧在30%以上,pH5.0,培养温度30℃,发酵时间48h;所述分批补料方法为:发酵12h后第一次补加培养基,并添加20%甘油,16h后第二次补加培养基,24h后第三次补加培养基,至发酵结束。超声破碎条件为:超声破碎频率为20kHz,破碎时间为20min,时间间隔为10:8s/s,气流干燥温度为28℃。
(7)NKEF重组蛋白在鲫鱼养殖中的应用
试验在6~9月份进行,将养殖鲫鱼网箱分成8组,5组网箱为试验组,3组为对照组,使用含NKEF的饲料(每公斤饲料添加含NKEF工程菌粉50mg)投喂,连续投喂8周,统计自然发病死亡率,计算相对免疫保护率(Relative Percent Survival, RPS),RPS=(1-免疫组死亡率/对照组死亡率)×100%。结果显示,实验组鲫鱼的病害发生率显著下降,平均成活率提高36.75%,相对免疫保护率RPS达86.47%,表明饲喂NKEF重组蛋白能增强鲫鱼在养殖中对病菌感染的抵抗力(表1);
表1 鲫鱼养殖抗病试验结果
(9)NKEF重组蛋白在鲈鱼养殖中的应用
试验在6~9月份进行,将养殖鲈鱼网箱分成8组,5组网箱为试验组,3组为对照组,使用含NKEF的饲料(每公斤饲料添加含NKEF工程菌粉50mg)投喂,连续投喂8周,统计自然发病死亡率,计算相对免疫保护率RPS。结果显示,实验组鲈鱼的发病率显著下降,平均成活率提高35.67%,相对免疫保护率RPS达87.94%,表明以饲喂NKEF重组蛋白能增强鲈鱼对病原菌感染的抵抗力(表2)。
表2 鲈鱼养殖抗病试验结果
本发明的操作步骤中的常规操作为本领域技术人员所熟知,在此不进行赘述。
以上所述的实施例对本发明的技术方案进行了详细说明,应理解的是以上所述仅为本发明的具体实施例,并不用于限制本发明,凡在本发明的原则范围内所做的任何修改、补充或类似方式替代等,均应包含在本发明的保护范围之内。
Claims (3)
1.一种鱼类天然杀伤细胞增强因子的酵母工程菌在制备提高鱼类存活率的饲料的应用,其特征在于:
所述酵母工程菌是INVSC1-pYES2-NKEF;
所述的酵母工程菌的构建方法包括以下步骤:
1)根据GeneBank数据库中登记的青斑河豚鱼NKEF基因序列Genbank No. DQ003333.1,设计表达载体构建引物P1和P2:
P1:5'-GGGAATTCAAAATGGCTGCAGGC-3';
P2:5'-CCGCTCGAGTTAGTGCTTGGAGAAG-3';
2)使用Trizol试剂盒提取河豚鱼脾脏总RNA,使用逆转录试剂盒将总RNA逆转录成cDNA模版;
3)以步骤2)得到的河豚鱼cDNA序列为模板,通过PCR扩增获得NKEF基因片段;PCR体系:10×PCR Buffer 2μL,dNTP Mixture 0.4μL,P1和P2各0.8μL,cDNA模版0.2μL,Ex Taqpolymerase 0.2μL,加ddH2O补足至20μL;
PCR参数:94℃预变性4min,94℃变性30s,58℃退火30s,72℃延伸30s,32个循环,72℃后延伸10min,15℃10min;
4)用限制性内切酶EcoRI和XhoI双酶切NKEF基因片段,纯化回收后与同样经双酶切处理的pYES2载体连接,转化大肠杆菌TOP10,获得到重组质粒pYES2-NKEF,选取阳性克隆;
5)将鉴定后的阳性克隆质粒电转化导入酿酒酵母INVSC1菌株中,获得酵母工程菌INVSC1-pYES2-NKEF;
6)将所述酵母工程菌INVSC1-pYES2-NKEF活化后接种至YPD培养基和发酵培养基中进行一级种和二级种摇瓶培养,取二级种培养物接种至50L全自动发酵罐中进行分批补料培养,激活其NKEF基因片段的表达,待所述酵母工程菌培养结束后收集菌体,用蒸馏水清洗后进行超声破碎处理,气流干燥制成酵母菌粉;
所述的发酵培养基成分及其重量份为:蔗糖90~120份、酵母粉20~35份、蛋白胨4~6份、尿素10~20份、麦芽汁2~4份、黄烯醇0.001~0.003份、KH2PO4 2~5份、K2HPO4·3H2O 2.0~3.5份、MgSO4·7H2O 0.06~0.2份、ZnSO4·7H2O 0.001~0.004份、MnSO4·4H2O 0.008~0.02份、木糖醇0.001~0.003份、生物素0.002~0.006份;
所述的一级种摇瓶培养条件为:培养温度25~35℃、摇床转速100~300r/min、培养时间18~24h;所述的二级种摇瓶培养条件为:接种量8~12%(V/V)、培养温度25~35℃、摇床转速100~300r/min、培养时间20~28h。
2.根据权利要求1所述的应用,其特征在于:所述的50L全自动发酵罐培养条件为:发酵培养基初始OD值为0.4~0.6,并补加1.4~2.5%半乳糖,溶氧在30%以上,pH4.5~5.5,培养温度25~35℃,发酵时间36~72h;所述分批补料方法为:发酵10~15h后第一次补加培养基,并添加15~25%甘油,14~18h后第二次补加培养基,22~26h后第三次补加培养基,至发酵结束。
3.根据权利要求1所述的应用,其特征在于:所述的超声破碎条件为:超声破碎频率为15~25kHz,破碎时间为17~23min,时间间隔为10:8s/s,气流干燥温度为25~30℃。
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