CN117089459A - 提高巴斯德毕赤酵母溶胞物溶出水平的方法 - Google Patents
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Abstract
本发明公开了一种提高巴斯德毕赤酵母溶胞物溶出水平的方法。所述方法先将表达重组人源胶原蛋白的巴斯德毕赤酵母Pichia pastoris JY0201高密度发酵液离心,获得的高密度菌液稀释后,加入促溶剂,调节pH为5~7,控制罐压为0.15~0.2MPa,并加热裂解,得到的自溶菌体混悬液经低温高速离心获得上清液,最后除菌过滤,得到巴斯德毕赤酵母提取液。本发明方法经济、高效,在常规溶出条件下,提高发酵罐压力,与常压相比,反应10h时氨基氮得率和酵母菌破壁率分别提高了85.901%、71.166%,溶出时间缩短16小时,且溶胞物具有优异的生物相容性。
Description
技术领域
本发明涉及一种提高巴斯德毕赤酵母溶胞物溶出水平的方法,属于微生物应用技术领域。
背景领域
酵母提取物来源于天然酵母,富含小分子氨基酸、肽、核苷酸、维生素等天然活性成分,其中氨基酸可为皮肤所利用,有利于胶原蛋白合成;肽作为信号分子参与多种生物反应,具有抗氧化作用;核酸及核苷酸促进新陈代谢、提高蛋白质合成速度、增强SOD活性,提高皮肤抗自由基能力;维生素C及其衍生物具备良好的美白作用。因此,酵母提取物具有显著改善皮肤状况的功效,包括减少经皮水分散失、改善皮肤粗糙度、降低黑色素、亮白皮肤、改善遗传皮肤过敏症状等,在化妆品领域具有广泛的应用前景。
为高效利用酵母胞内营养,充分获得酵母提取物,人们研发了多种破壁方法,包括高压均质法、超声波法、酶法、自溶法、脉冲电场等。其中,自溶法是利用酵母的酶活,在特定温度和pH值下细胞通过自身含有的水解酶将多糖、蛋白质和核酸等大分子物质分解成可溶的小分子如还原糖、氨基酸和核苷酸等,其具有以下优点:利用酵母自身酶系进行蛋白质分解,蛋白质分解率较其他方法高,游离氨基酸含量高,成本低。但由于酵母细胞内源酶活力有限,常借助一些物理、化学和生物手段来提高自溶率。例如,添加适量的氯化钠或乙醇等促溶剂,使酵母质壁分离,细胞膜功能失调,胞内水解酶溶出,加速自溶过程再促使自溶。
现今酵母提取液的制备主要以酿酒酵母、啤酒酵母等为原料,有关表达重组胶原蛋白的巴斯德毕赤酵母发酵菌利用的相关报道极少。巴斯德毕赤酵母表达系统在多种类型的外源蛋白表达上优势十分明显,具有易于达到高密度发酵、产物高表达的特点。重组胶原蛋白在巴斯德毕赤酵母中的表达为分泌表达,蛋白可通过低温高速离心等方式与酵母菌体实现快速分离,分离后菌渣液中仍残留少量胶原蛋白。高密度发酵产生了大量的酵母菌渣液,通常占据整个发酵液体积的35%左右,需灭活后经环保部门处理方能达到国家排放标准。对于规模化发酵分离出的数量庞大的巴斯德毕赤酵母菌体,需要合理有效地利用发酵系统资源基础上,尽可能减少设备占用,采取简单高效的处理方法,降低长时间存储和反应时间长引起的变质问题。
发明内容
本发明的目的在于提供一种提高巴斯德毕赤酵母溶胞物溶出水平的方法。该方法通过提高发酵罐压力至0.15~0.2MPa,缩短了溶出时间,且提高了胞内营养物溶出度。
实现本发明目的的技术方案如下:
提高巴斯德毕赤酵母溶胞物溶出水平的方法,通过将高密度发酵液依次经过稀释、加热裂解、低温高速离心、除菌过滤处理,获得巴斯德毕赤酵母提取液,具体步骤如下:
(1)将表达重组人源胶原蛋白的巴斯德毕赤酵母Pichiapastoris JY0201高密度发酵液离心,获得的高密度菌液通入发酵罐中,加入无菌纯化水稀释成菌悬液后,加入促溶剂,调节pH为5~7,控制发酵罐压力为0.15~0.2MPa,并加热至45~80℃,裂解得到自溶菌体混悬液;
(2)将自溶菌体混悬液在10~12℃下低温高速离心多次,获得澄清透明上清液;
(3)将上清液除菌过滤,得到巴斯德毕赤酵母提取液。
本发明所述的表达重组人源胶原蛋白的巴斯德毕赤酵母PichiapastorisJY0201,其保藏编号为CGMCC No.16461,已在中国专利201811589560.X公开。
优选地,步骤(1)中,菌悬液的湿重为235g/L~351g/L,更优选为260g/L~280g/L。
优选地,步骤(1)中,促溶剂为氯化钠,添加量为0.02~0.06g/mL。
优选地,步骤(1)中,加热裂解时间为8~48h。
在本发明具体实施方式中,步骤(1)中,氯化钠的添加量为0.036g/mL,调节pH=5.4,加热温度为60℃,加热裂解时间为16h。
优选地,步骤(2)中,高速离心的转速为8000g~14000g,离心次数为8~9次。
优选地,步骤(3)中,除菌过滤的具体方法为将上清液先后经0.45μm、0.22μm孔径的PES滤膜进行二级过滤。
与现有技术相比,本发明具有以下优点:
本发明在常规溶出条件下,提高发酵罐压力至0.15~0.2MPa,使得氨基氮得率和酵母菌破壁率分别提高了85.901%、71.166%,明显提高了巴斯德毕赤酵母溶胞物溶出水平。采用本发明提取方法获得的巴斯德毕赤酵母提取液中含有0.62~1.56g/L重组人源I型胶原蛋白,其稀释80倍(1.25%)后,在L929鼠成纤维细胞实验中未检测出潜在细胞毒性,与HFF-1、HaCat表皮细胞反应10小时、巴斯德毕赤酵母溶胞物浓度为1%时,细胞增殖能力最佳,表明巴斯德毕赤酵母溶胞物具有优异的生物相容性。
附图说明
图1为细胞毒性实验图。
图2为HaCat表皮细胞增殖实验图。
图3为HFF-1成纤维细胞增殖实验图。
具体实施方式
下面结合具体实施例、对比例和附图对本发明作进一步详述。
下述实施例和对比例中,采用的巴斯德毕赤酵母为表达重组人源胶原蛋白的巴斯德毕赤酵母Pichia pastoris JY0201,保藏编号为CGMCC No.16461,已在中国专利201811589560.X充分公开。
实施例1
(1)将巴斯德毕赤酵母Pichiapastoris JY0201高密度发酵液离心,获得的高密度菌液用无菌纯化水稀释至酵母菌体湿重为278g/L的菌悬液。
(2)在稀释后的菌悬液中加入NaCl,使得NaCl的浓度为0.036g/mL,调节pH至5.4,设定搅拌轴转速200rpm,发酵罐压力为0.15MPa,夹套蒸汽加热循环至60℃维持并计时,维持时间共计16h,裂解获得自溶菌体混悬液。
(3)自溶菌体混悬液泵入碟式离心机离心,控温10~12℃,离心力14000g,循环离心9次,获得澄清透明上清液。
(4)将上清液输入储罐,由管路输送至洁净区,高压空气下,先后经0.45μm和0.22μmPES膜进行二级过滤,无菌灌装获得巴斯德毕赤酵母Pichiapastoris JY0201提取液。
实施例2
本实施例与实施例1基本相同,唯一不同的是(2)中发酵罐压力为0.20MPa。
对比例1
(1)将巴斯德毕赤酵母Pichiapastoris JY0201高密度发酵液离心,获得的高密度菌液用无菌纯化水稀释至酵母菌体湿重为278g/L的菌悬液。
(2)在稀释后的菌悬液中加入3.6%NaCl(W/V),调节pH至5.4,设定搅拌轴转速200rpm,不调节罐压,夹套蒸汽加热循环至60℃维持并计时,维持时间共计16h,裂解获得自溶菌体混悬液。
(3)自溶菌体混悬液泵入碟式离心机离心,控温10~12℃,离心力14000g,循环离心9次,获得澄清透明上清液。
(4)将上清液输入储罐,由管路输送至洁净区,高压空气下,先后经0.45μm和0.22μmPES膜进行二级过滤,无菌灌装获得巴斯德毕赤酵母Pichiapastoris JY0201提取液。
对比例2
本对比例与实施例1基本相同,唯一不同的是(2)中发酵罐压力为0.03MPa。
对比例3
本对比例与实施例1基本相同,唯一不同的是(2)中发酵罐压力为0.05MPa。
对比例4
本对比例与实施例1基本相同,唯一不同的是(2)中发酵罐压力为0.10MPa。
性能检测例
1.对各实施例和对比例获得的溶胞物进行检测,具体检测方法如下:
(1)毕赤酵母菌破壁率
自溶菌体混悬液按照固定比例稀释,与美兰染色液1:1混合均匀染色,采用血球计数板法,记录细胞总数和活细胞数,根据下述公式计算毕赤酵母破壁率:
毕赤酵母破壁率(%)=(细胞总数-活细胞数)/细胞总数×100%。
(2)氨基氮得率
氨基氮质量浓度的测定采用甲醛滴定法,记录加热裂解处理不同时间点获得的上清中氨基氮质量浓度以及加热裂解前菌悬液中氨基氮质量浓度,根据下述公式计算氨基氮得率:
氨基氮得率(%)=(加热裂解后获得的上清中氨基氮质量浓度/加热裂解前菌悬液中氨基氮质量浓度)×100%。
表1各实施例、对比例在不同时间点的氨基氮得率和破壁率
从表1可以看出,对比例2、对比例3与对比例1相比,氨基氮得率与破壁率略有提高,但不明显。对比例4将发酵罐压力提高至0.1MPa,反应8小时,氨基氮得率、破壁率有较大幅度提升,而实施例1和实施例2将发酵罐压力提高至0.15~0.2MPa,氨基氮得率和破壁率分别提高了85.901%、71.166%。
2.细胞毒性和细胞增殖能力检测
选取实施例2裂解不同时间段的酵母溶胞物和对比例1裂解26h的酵母溶胞物,经0.22μm PES膜除菌过滤,对其细胞毒性和细胞增殖能力进行检测。具体如下:
(1)细胞毒性试验-MTT
全程在超净台内无菌操作。取处于对数生长期的L929细胞,用胰蛋白酶消化,并将细胞悬浮液离心(1000rpm,5min)后弃去原培养物,然后重悬于培养基中,得到1×105个/mL细胞悬液。
将悬浮细胞以每孔100μL接种于96孔板中,并在细胞培养箱(37℃,5%CO2)中培养24小时。镜下观察细胞形态。
细胞生长形成单层后,弃去96孔板内原培养基。在96孔板相应孔内各加入100μL样品,空白对照样品和阳性对照样品(1%ZDEC)。将96孔板置于细胞培养箱(37℃,5%CO2)中培养24小时,每组5个复孔。
培养24小时后取出96孔板,在显微镜下观察细胞形态,接着去除液体,每孔加入50μLMTT(1mg/mL),置二氧化碳培养箱中培养。2小时后去除上清,每孔加入100μLDMSO溶解结晶,在酶标仪上测定570nm波长下的吸光度值,计算其细胞毒性。
结果如图1所示,作用于L929鼠成纤维细胞时,酵母溶胞物在5%、2.5%、1.5%浓度下均未显现潜在细胞毒性。
(2)成纤维细胞(HFF-1)、表皮细胞(HaCat细胞)细胞增殖试验-MTT法
全程在超净台内无菌操作。取处于对数生长期的HFF-1细胞(HaCat细胞),用胰蛋白酶消化,并将细胞悬浮液离心(1000rpm,5min)后弃去原培养物,然后重悬于培养基中,得到细胞悬液。
将悬浮细胞以每孔100μL接种于96孔板中,并在细胞培养箱(37℃,5%CO2)中培养24小时。镜下观察细胞形态。
细胞生长形成单层后,弃去96孔板内原培养基。在96孔板相应孔内各加入100μL样品,空白对照样品。将96孔板置于细胞培养箱(37℃,5%CO2)中培养48小时,每组5个复孔。
培养24小时后取出96孔板,在显微镜下观察细胞形态,然后去除液体,每孔加入50μLMTT(1mg/mL),置二氧化碳培养箱中培养。2小时后去除上清,每孔加入100μLDMSO溶解结晶,于酶标仪检测,选择双波长检测,检测波长为490nm,以630nm为参考波长,最终OD值为490nm的OD值减去630nm的OD值,计算其细胞毒性。
结果如图2和3所示,对比例1和实施例2的酵母溶胞物都能促进HaCat、HFF-1细胞增殖,其中实施例2组中溶出时间为10h、1%浓度下增殖能力最强;在相同浓度下,实施例2-8h样品细胞增殖能力略优于对比例1-26h,说明长时间反应对溶胞物的组成有一定影响。
Claims (8)
1.提高巴斯德毕赤酵母溶胞物溶出水平的方法,其特征在于,具体步骤如下:
(1)将表达重组人源胶原蛋白的巴斯德毕赤酵母Pichia pastoris JY0201高密度发酵液离心,获得的高密度菌液通入发酵罐中,加入无菌纯化水稀释成菌悬液后,加入促溶剂,调节pH为5~7,控制发酵罐压力为0.15~0.2MPa,并加热至45~80℃,裂解得到自溶菌体混悬液;
(2)将自溶菌体混悬液在10~12℃下低温高速离心多次,获得澄清透明上清液;
(3)将上清液除菌过滤,得到巴斯德毕赤酵母提取液。
2.根据权利要求1所述的方法,其特征在于,步骤(1)中,菌悬液的湿重为235g/L~351g/L。
3.根据权利要求1所述的方法,其特征在于,步骤(1)中,菌悬液的湿重为260g/L~280g/L。
4.根据权利要求1所述的方法,其特征在于,步骤(1)中,促溶剂为氯化钠,添加量为0.02~0.06g/mL。
5.根据权利要求1所述的方法,其特征在于,步骤(1)中,加热裂解时间为8~48h。
6.根据权利要求1所述的方法,其特征在于,步骤(1)中,氯化钠的添加量为0.036g/mL,调节pH=5.4,加热温度为60℃,加热裂解时间为16h。
7.根据权利要求1所述的方法,其特征在于,步骤(2)中,高速离心的转速为8000g~14000g,离心次数为8~9次。
8.根据权利要求1所述的方法,其特征在于,步骤(3)中,除菌过滤的具体方法为将上清液先后经0.45μm、0.22μm孔径的PES滤膜进行二级过滤。
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