CN113005154B - 一种提高裂殖壶菌中二十碳五烯酸产量的方法 - Google Patents
一种提高裂殖壶菌中二十碳五烯酸产量的方法 Download PDFInfo
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Abstract
本发明公开了一种提高裂殖壶菌中二十碳五烯酸产量的方法,将裂殖壶菌(Schizochytrium sp.)ATCC 20888接种在发酵培养基中,在有氧条件下进行发酵,发酵至对数期中期时进行变温,并继续发酵,且变温后控制溶氧值(DO)在2%~10%,所述变温为将起始发酵温度25~30℃提高至32~37℃。以裂殖壶菌Schizochytrium sp.ATCC 20888发酵生产EPA,得到的发酵液中菌体干重达到66.15g/L,油脂产量为9.97g/L,EPA占脂肪酸的百分含量达到13.33%。本发明能应用于大规模的裂殖壶菌发酵,变温及低溶氧的培养方式能在工厂中有效实施,最大限度提高了底物利用效率,并且提高产物中的二十碳五烯酸的含量。
Description
技术领域
本发明属于发酵领域,涉及一种提高裂殖壶菌中二十碳五烯酸产量的方法。
背景技术
长链多不饱和脂肪酸(LC-PUFA)是指具有两个或两个以上双键,碳链长度为18-22个碳原子的直链脂肪酸。LC-PUFA与维生素、矿物质元素一样,是人体必需的营养素,是一种具有重要医药保健作用的物质。LC-PUFAs可分为ω3和ω6多不饱和脂肪酸。在ω3多不饱和脂肪酸中,二十二碳六烯酸(DHA)和二十碳五烯酸(EPA)是最重要的。ω3多不饱和脂肪酸有突出效果保持心脏的健康功能,心血管、肾脏和大脑,预防肥胖代谢综合征、心血管疾病、炎症、神经退行性疾病和其他疾病。长期摄入不足容易导致心、脑等重要器官功能障碍。ω3多不饱和脂肪酸的全球需求将逐年增加,人民生活水平的不断提高,追求一个更健康的生活。据估计,从2015年到2025年,全球需求的ω3多不饱和脂肪酸每年将增长16%。99%的DHA市场价格为144美元/克,而99%的EPA市场价格为2000美元/克,远远高于DHA。
二十碳五烯酸(EPA)是一种对人体极为重要的ω3多不饱和脂肪酸,对于心血管疾病的防治、精神分裂症、抑郁症的治疗及抗炎抗癌等方面具有重要的生理功能,目前,EPA在保健食品、药品、饲料等行业具有广阔的商业前景。EPA的传统来源是鱼油,但由于受到海洋资源的告竭和环境污染等因素的影响,鱼油的质量和产量不尽人意,无法满足人们日益增长的需求,寻找一条可持续生产的绿色途径是目前亟待解决的问题。
Schizochytrium sp.是一种异养海洋真菌,含有大量DHA(40-60%),EPA同样存在其脂肪酸中,但通常EPA占总脂肪酸(TFAs)的百分含量低于1%,所以裂殖壶菌主要用于发酵生产DHA。裂殖壶菌生长速度快、产量高,常被认为是一种令人满意的DHA生产可持续资源,并且是世界各国批准可用于商业化生产DHA的微藻之一,具有很大的商业前景。由于EPA在裂殖壶菌中含量较少,目前关于裂殖壶菌的研究主要集中于优良菌株选育、DHA的生物合成及发酵条件优化等方面,关于利用裂殖壶菌发酵制备EPA的研究较少。
凌雪萍等人通过在裂殖壶菌培养至24h时添加50mg/L氟啶酮从而使EPA占总脂肪酸的百分含量从0.45%提高至0.65%。(凌雪萍,李俊,卢英华等.一种提高裂殖壶菌中EPA含量的调控方法与应用:.)孟彤通过发酵过程中补料分批发酵及过程添加无机盐发酵168h,使EPA占总脂肪酸的百分含量从0.58%提升至0.98%。(孟彤.裂殖壶菌生产ω-3多不饱和脂肪酸发酵技术研究[D].厦门大学,2019.)
目前对于裂殖壶菌产EPA的研究都存在EPA占总脂肪酸百分含量低,发酵时间长,外源添加物具有毒性等问题,因此EPA由裂殖壶菌发酵生产代替从鱼油中提取的关键在于通过优化发酵工艺进一步提高EPA的产量。
发明内容
针对现有的发酵技术基础进行改进,本发明提供了一种提高裂殖壶菌中二十碳五烯酸产量的方法,目的在于通过改变发酵温度温和控制溶氧的方法提高裂殖壶菌产物中二十碳五烯酸的合成量,进一步提高二十碳五烯酸的产量。
本发明的目的通过以下技术方案实现:
1)菌株:裂殖壶菌(Schizochytrium sp.ATCC 20888)购自美国典型培养物保藏中心。
2)培养基:
固体培养基(g/L)为葡萄糖30~50、酵母浸粉8~10、谷氨酸钠10~30、硫酸镁3~5、硫酸铵1.5~3、硫酸钠20~40、磷酸二氢钾2~5、氯化钾0.5~1.5、微量元素溶液1.5~3mL、琼脂粉15-20。
种子培养基(g/L)包括葡萄糖50~80、酵母浸粉8~10、谷氨酸钠40~80、硫酸镁3~5、硫酸铵1.5~3、硫酸钠20~40、磷酸二氢钾2~5、氯化钾0.5~1.5,微量元素溶液1.5~3mL。
发酵培养基(g/L)包括葡萄糖100~120、酵母浸粉5~8、谷氨酸钠20~60、硫酸镁3~5、硫酸铵3~5、硫酸钠20~40、磷酸二氢钾2~5、氯化钾0.5~1.5、微量元素溶液1.5-3mL。
3)微量元素溶液(g/L)的配方为EDTA二钠5~8、氯化钴0.005~0.02、氯化锰0.5~1、硫酸锌1~3、硫酸亚铁0.05~1、硫酸铜0.5~1、钼酸钠0.005~0.02、硫酸镍0.05~1。
4)细胞密度:紫外分光光度计,600nm波长。取样品适当稀释,测定范围0.2-0.8,计算时稀释倍数乘测定值。重复三次。
5)DCW测定:取10mL发酵液,5000g离心5min倒掉上清,去离子洗涤菌体两次,获得裂殖壶菌湿菌体。将湿菌体置于80℃烘箱中烘干,烘干后称取干菌体重量至恒重。重复三次。
6)总脂的测定:取10mL发酵液5000g离心5min,去离子水洗涤2次。湿菌体加5mL盐酸,涡旋2min,置于80℃水浴锅水浴加热1h,正己烷萃取3次直至上清液透明。油样全部溶解在正己烷溶液中,旋蒸回收溶剂,烘干溶剂,称量油脂。用3mL 2%氢氧化钠甲醇进行60℃、30min的甲酯化操作,得到的脂肪酸甲酯采用气相色谱-质谱联用仪进行检测。气相色谱柱为CP-SiL88,使用的载气为氦气,采用的进样方式为分流进样,色谱柱的升温程序为:初始温度140℃,保持5min后以10℃/min的速度升温至220℃,保持17min。根据内标采用峰面积归一化法计算总脂和各脂肪酸含量。
将裂殖壶菌(Schizochytriumsp.)ATCC 20888接种在发酵培养基中,在有氧条件下进行发酵,发酵至对数期中期时进行变温,并继续发酵,且变温后控制溶氧值(DO)在2%~10%,所述变温为将起始发酵温度25~30℃提高至32~37℃。
优选的,对数期中期对应的发酵时间为24±4h,所述起始发酵温度为28±1℃,变温后的温度34±1℃。
优选的,变温前的溶氧值(DO)控制在50%以上。
优选的,发酵培养基中含有微量元素溶液(g/L)的配方为:EDTA二钠5~8、氯化钴0.005~0.02、氯化锰0.5~1、硫酸锌1~3、硫酸亚铁0.05~1、硫酸铜0.5~1、钼酸钠0.005~0.02、硫酸镍0.05~1。
优选的,发酵培养基(g/L)的组成为:葡萄糖100、酵母浸粉8、谷氨酸钠40、硫酸镁4.48、硫酸铵1.5、硫酸钠37、磷酸二氢钾3.5、氯化钾1、微量元素溶液2mL。
优选的,发酵过程中保证葡萄糖含量维持于10-30g/L。
优选的,变温发酵反应的时间为96h±4h。
优选的,发酵条件为:pH值5.5~7,通气3~5L/min,转速为300~700rpm。
本发明与现有技术相比,具有如下有益效果:
通过建立一种提高裂殖壶菌中二十碳五烯酸产量的方法,在发酵对数中期升高培养温度及低溶氧控制的方法,提高了裂殖壶菌中EPA占总脂肪酸的百分含量,油脂产量达到9.97g/L,EPA占总脂肪酸百分含量达到13.33%,使裂殖壶菌油脂品质得到提升。
附图说明
图1为28℃发酵裂殖壶菌生长特性。
图2为34℃发酵裂殖壶菌生长特性。
图3为10%DO及2%DO条件下发酵裂殖壶菌生长特性,图3a为发酵裂殖壶菌生长特性;图3b为发酵裂殖壶菌的油脂产量;图3c为发酵裂殖壶菌谷氨酸钠的消耗情况;图3d为发酵裂殖壶菌DHA、EPA占总脂肪酸百分含量。
具体实施方式
下面结合具体实施例对本发明作进一步具体详细描述,但本发明的实施方式不限于此,对于未特别注明的工艺参数,可参照常规技术进行。
实施例1
考察裂殖壶菌在适宜生长温度(28℃)和高温(34℃)条件下的生长特性,分别在5L发酵罐进行两种温度条件的发酵。
将摇好的裂殖壶菌种子液按10%接种量接种至5L发酵罐中,培养温度分别为28℃和34℃,pH自然,通气3L/min,转速为500rpm,葡萄糖含量低于20g/L开始补加葡萄糖,使葡萄糖含量维持于20g/L以上,发酵时间为120h。
结果表明,28℃比较适宜裂殖壶菌的生长,发酵结束时生物量达到63.31g/L,油脂产量达到20.39g/L,EPA占总脂肪酸的百分含量为0.86%(表1);在34℃发酵时,裂殖壶菌生长受限,发酵至120h生物量为31.34g/L,油脂产量为3.97g/L,但EPA占总脂肪酸的百分含量显著增加,发酵结束时EPA占总脂肪酸的百分含量达到7.17%(表1)。
表1 28℃和34℃条件下裂殖壶菌的脂肪酸组成变化情况
实施例2
考察溶氧条件对裂殖壶菌,分别探究不同溶氧(DO)(10%和2%)条件的生长情况,分别在5L发酵罐进行两种溶氧条件的优化。
将摇好的裂殖壶菌种子液按10%接种量接种至5L发酵罐中,培养温度为28℃,pH自然,通气3L/min,初始转速为500rpm,待溶氧降至50%后,调整转速和通气维持前24h溶氧值(DO)维持在50%,后96h分别维持在10%和2%,葡萄糖含量低于20g/L开始补加葡萄糖,使葡萄糖含量维持于20g/L以上,发酵时间为120h。
不同溶氧条件下裂殖壶菌生长特性如图3所示。与2%DO条件相比,10%DO条件下的细胞生长较好,生物量最高达到75.067g/L(图3a),在36h时谷氨酸钠消耗较快(图3c),但油脂产量(图3b)、DHA占总脂肪酸百分含量和EPA占总脂肪酸百分含量低于2%DO(图3d)。在2%DO条件下裂殖壶菌的生物量在发酵过程中继续上升,最高达到61.21g/L,油脂产量和EPA占总脂肪酸百分含量分别达到20.39g/L和3.29%。结果表明,细胞在高溶氧水平下生长较好,而低溶氧水平对油脂和EPA的积累有正向影响。
实施例3
不同变温时间节点优化:考察变温时间节点对裂殖壶菌,分别探究不同变温时间(24h、48h、72h)条件的生长情况,分别在5L发酵罐进行三种变温时间节点的优化。实验分为24h变温组、48h变温组和72h变温组,分别在发酵至24h(对数期中期)、48h(稳定期前期)及72h(稳定期中期)进行变温,温度升高至34℃,28℃恒温发酵为对照组,将摇好的裂殖壶菌种子液按10%接种量接种至5L发酵罐中,培养温度分别为28℃,pH自然,通气3L/min,转速为500rpm。发酵结果如表2所示。
表2不同变温时间对裂殖壶菌的生物量、油脂产量、EPA的百分含量及EPA产量的影响
对照组 | 24h变温组 | 48h变温组 | 72h变温组 | |
生物量(g/L) | 63.31 | 43.17 | 53.83 | 68.44 |
油脂产量(g/L) | 20.39 | 10.23 | 10.55 | 20.10 |
EPA含量(%TFAs) | 0.86 | 7.93 | 4.93 | 2.31 |
EPA产量(g/L) | 0.17 | 0.78 | 0.50 | 0.46 |
结果发现变温时间越晚,对裂殖壶菌的生长影响越小,在72h进行变温,裂殖壶菌的生长基本不受影响,在油脂积累方面,高温条件会减少裂殖壶菌油脂积累,在24h变温发酵结束时EPA含量(%TFAs)最高,EPA占总脂肪酸百分含量为7.93%,与恒温发酵相比提高了9.22倍,综上所述,24h变温为最优条件。
实施例4
裂殖壶菌在28℃条件下,在对数中期(24h)进行变温,温度升高至34℃,初始通气量为3L/min,初始转速为500rpm,pH自然,待溶氧降至50%后,调整转速和通气维持溶氧在前24h于50%,后96h维持溶氧在2%,高温与低溶氧条件诱导裂殖壶菌积累EPA。将实验结果与实施例1中的数据进行比较,见表4
结果表明,由表3可知,在该策略条件下,前期发酵温度控制在28℃可以保证细胞密度高,发酵至24h时调高温度至34℃,促进EPA积累,EPA占总脂肪酸的百分含量显著提高,发酵结束时EPA的百分含量提高至13.33%。
表3变温条件下裂殖壶菌生物量、油脂产量及几种主要脂肪酸的变化情况
由表4可知,在此优化工艺下进行发酵,裂殖壶菌的生物量及EPA占总脂肪酸的百分含量显著高于在普通发酵工艺,阶段控温及低溶氧策略下裂殖壶菌发酵120h时生物量达到66.15g/L,油脂产量达到9.97g/L,EPA占总脂肪酸百分含量达到13.33%。
表4裂殖壶菌在不同温度控制策略下EPA发酵相关参数的比较
a:与实施例1中28℃和34℃发酵条件下的最佳结果进行比较。
上述实施例为本发明较佳的实施方式,但本发明的实施方式并不受上述实施例的限制,其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化,均应为等效的置换方式,都包含在本发明的保护范围之内。
Claims (6)
1.一种提高裂殖壶菌中二十碳五烯酸产量的方法,其特征在于,将裂殖壶菌(Schizochytriumsp.)接种在发酵培养基中,在有氧条件下进行发酵,发酵至对数期中期时进行变温,并继续发酵,且变温后控制溶氧值在2%~10%,所述变温为将起始发酵温度28±1℃提高至34±1℃;
所述对数期中期对应的发酵时间为24±4h;
所述变温前的溶氧值控制在50%以上,所述裂殖壶菌(Schizochytriumsp.)为裂殖壶菌(Schizochytriumsp.)ATCC 20888。
2.根据权利要求1所述的一种提高裂殖壶菌中二十碳五烯酸产量的方法,其特征在于,所述发酵培养基中含有的微量元素溶液的配方为:EDTA二钠5~8 g/L、氯化钴0.005~0.02g/L、氯化锰0.5~1 g/L、硫酸锌1~3 g/L、硫酸亚铁0.05~1 g/L、硫酸铜0.5~1 g/L、钼酸钠0.005~0.02 g/L、硫酸镍0.05~1 g/L。
3.根据权利要求2所述的一种提高裂殖壶菌中二十碳五烯酸产量的方法,其特征在于,所述的发酵培养基的组成为:葡萄糖100 g/L、酵母浸粉8 g/L、谷氨酸钠40 g/L、硫酸镁4.48 g/L、硫酸铵1.5 g/L、硫酸钠37 g/L、磷酸二氢钾3.5 g/L、氯化钾1 g/L、微量元素溶液2 mL/L。
4.根据权利要求1或2所述的一种提高裂殖壶菌中二十碳五烯酸产量的方法,其特征在于,发酵过程中保证葡萄糖含量维持于10-30 g/L。
5.根据权利要求1或2所述的一种提高裂殖壶菌中二十碳五烯酸产量的方法,其特征在于,所述变温后发酵的时间为96h±4h。
6.根据权利要求1或2所述的一种提高裂殖壶菌中二十碳五烯酸产量的方法,其特征在于,所述发酵条件为:pH值5.5~7,通气3~5L/min,转速为300~700rpm。
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