CN112877378B - 一种提高裂殖壶菌发酵生产油脂中奇数碳脂肪酸产量的方法 - Google Patents
一种提高裂殖壶菌发酵生产油脂中奇数碳脂肪酸产量的方法 Download PDFInfo
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Abstract
一种提高裂殖壶菌发酵生产油脂中奇数碳脂肪酸产量的方法,将裂殖壶菌(Schizochytrium sp.)27173‑E接种在发酵培养基中,进行低温有氧发酵,控制培养基中的总碳源和氮源质量比为5:1~20:1,温度在15℃~35℃,发酵时间为60~120h。裂殖壶菌27173‑E发酵生产OCFA的最优化条件是:总碳源和氮源质量比为10:1,有机氮与无机氮比为8:1,发酵温度为23℃,发酵时间84h。应用此方法,得到的发酵液中细胞最大干重达到78.87g/L,OCFA占总脂肪酸的31.82%,OCFA产量为7.48g/L。本发明极大提高了OCFA的产量,进一步扩大了裂殖壶菌的应用潜力。
Description
技术领域
本发明属于微生物发酵领域,具体涉及一种提高裂殖壶菌发酵生产油脂中奇数碳脂肪酸产量的方法。
背景技术
奇数碳脂肪酸(Odd chain fatty acid,OCFA)的功能类似不饱和脂肪酸,有助于提高细胞膜的流动性。在某些细菌中含量较多,但在动物、低等植物中含量不超过1%。奇数碳脂肪酸代谢后产生糖残基,因而具有更佳的营养效果;此外,含有奇数碳脂肪酸的甘油三酯可以减少脓毒症及重症特护治疗的继发性并发症的发生频率,或减轻炎症性或缩短病程(CN200980145702.6)。近些年的研究发现在人体中含量较少的奇数碳链脂肪酸,具有重要的生理功能,有研究显示人体中奇数碳链脂肪酸含量与糖尿病、心血管疾病、肥胖等都呈负相关。
动植物油脂中绝大多数脂肪酸的碳链长度为偶数,少数为奇数,比如十五酸、十七酸。奇数碳脂肪酸主要来自反刍动物的瘤胃细菌,主要存在于动物乳脂中,C15:0和C17:0分别约占到总脂肪酸的1.2%和0.54%。奇数碳脂肪酸可通过偶数碳脂肪酸的α氧化,以及脂肪酸合成两条途径产生。α氧化一般发生在脂肪酸β位存在基团修饰的情况下,正常情况下细胞内不会大量积累这种脂肪酸。目前奇数碳链的脂肪酸产品并未问世,其主要原因是奇数碳链脂肪酸来源很少,目前的改良途径主要有两个,一方面通过添加丙酸盐,Zhang等人在红浊球菌发酵培养基中添加1%-1.5%异丙醇,最终OCFA占总脂肪酸比例提高至50%。另一方面,通过添加OCFA合成的前体物质来增加其产量(CN201280054084.6)。此专利通过增强丙酰辅酶A的合成来提高OCFA的产量,但OCFA的总量只有0.325g/L。
微生物是生产奇数碳链脂肪酸的较好来源,裂殖壶菌又称裂壶藻,属于破囊壶菌科的一类海洋真菌。裂殖壶菌能够积累大量活性物质,如DHA,DPA,胡萝卜素,角鲨烯等。采用葡萄糖或甘油做碳源发酵,细胞干重可达到150g/L,油脂占细胞干重能到70%以上。裂壶藻有很强的脂质积累能力和较纯的脂肪酸组成,是极佳的脂质合成宿主。裂壶藻可用于生产奇数碳脂肪酸,Chang等介绍了破囊壶菌科Schizochytrium疑似菌株积累15.4%的OCFA。EP0823475A中,Schizochytrium SR21菌奇数碳脂肪酸中C15:0为10.1%,C17:0为1.8%。Chang K等将裂壶藻ATCC20888分批发酵,在发酵72h时检测到最高量的C15:09.16%,C17:0为2.91%。(Chang K,Mansour M P,Dunstan G A,et al.Odd-chain polyunsaturatedfatty acids in thraustochytrids[J].Phytochemistry,2011,72(11-12):1460-1465.)Schizochytrium藻发酵生产奇数碳脂肪酸,细胞内最高含量的报道为12.07%。
裂壶藻发酵产脂的报道已经很多,但微生物发酵产OCFA,需要丙酸盐诱导,生物量低,油脂产量低,OCFA含量低。而基因改造成本高,大多处于理论研究阶段,现有文献报道的通过基因工程获得的OCFA最高值3.32g/L。本发明能够克服现有技术缺陷,可提供一种提高裂殖壶菌油脂中奇数碳链脂肪酸含量的方法。
发明内容
为了克服现有技术存在的上述不足,本发明的目的是提供一种提高裂殖壶菌发酵生产油脂中奇数碳脂肪酸产量的方法,旨在提高裂殖壶菌油脂产物中奇数碳链脂肪酸的百分含量,提高生产效率。
将裂殖壶菌(Schizochytrium sp.)接种在发酵培养基中,进行低温有氧发酵,控制培养基中的总碳源和氮源质量比为5:1~20:1,温度在15℃~35℃,发酵时间为60~120h。
优选的,培养基中的总碳源和氮源质量比为10:1。
优选的,培养基中有机氮N1与无机氮N2质量比为1:1~9:1。
优选的,培养基中有机氮N1与无机氮N2质量比为8:1。
优选的,发酵温度为23±2℃,发酵时间为84±12h。
优选的,培养条件为:pH为7~8,通气为3vvm,转速600rpm。
优选的,发酵培养基的组分为,每1L培养基中,葡萄糖20-80g,氯化钾0.5-1.5g,酵母粉5-10g,七水合硫酸镁4-8g,磷酸二氢钾3-5g,无水硫酸钠20-40g,谷氨酸钠5-40g,硫酸铵1-6g,金属离子溶液2-3mL,消泡剂0.0005-0.001g。
优选的,金属离子溶液配方(g/L):CoCl2·6H2O 0.2-0.5;CuCl2·2H2O0.5-1.0;Na2·EDTA 2.0-10.0;H3BO32.0-5.0;MnCl2·H2O 1.0-5.0;NiCl·6H2O 0.5-1.0;ZnSO4·7H2O 2.0-5.0;NaMoO4·H2O 1.0-3.0。
优选的,裂殖壶菌(Schizochytrium sp.)为裂殖壶菌(Schizochytrium sp.)27173-E。
本发明与现有技术相比,具有如下有益效果:
本发明建立了一种提高裂殖壶菌发酵生产油脂中奇数碳脂肪酸产量的方法,将培养基组分中总碳氮质量比控制在10:1,有机氮与无机氮质量比调整为8:1,温度条件为23℃。最终在5L发酵放大,生物量在发酵终点最高达到78.87g/L,油脂产量在发酵96h达到最高值24.95g/L,OCFA占总脂肪酸的比例在发酵84h达到32.19%,OCFA产量在发酵84h达到最高产量7.48g/L。相比于裂殖壶菌目前通过基因工程获得的最大产量3.32g/L,提高了125%。
附图说明
图1为细胞生长随时间变化图。
图2为油脂积累随时间变化图。
图3为奇数碳脂肪酸积累随时间变化图。
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。
1)菌株:裂殖壶菌(Schizochytrium sp)27173-E购自美国典型培养物保藏中心,ATCC登录号PTA-9695,公开于公开号为CN102428185,US8207363的专利申请文件中。
2)培养基:
斜面培养基(1L):葡萄糖80g,氯化钾1g,酵母粉10g,七水合硫酸镁4.5g,磷酸二氢钾3.5g,无水硫酸钠37g,谷氨酸钠40g,琼脂20g,蒸馏水定容至1000mL;
种子培养基(1L):葡萄糖80g,氯化钾1g,酵母粉10g,七水合硫酸镁4.5g,磷酸二氢钾3.5g,无水硫酸钠37g,谷氨酸钠40g,金属离子溶液2mL,蒸馏水定容至1000mL;
发酵培养基(1L):葡萄糖80g,氯化钾1g,酵母粉5g,七水合硫酸镁4.5g,磷酸二氢钾3.5g,无水硫酸钠37g,谷氨酸钠40g,硫酸铵1.2g,金属离子溶液2mL,消泡剂0.0005g,蒸馏水定容至1000mL。
消泡剂为道康宁1520消泡剂,购自深圳市宝安区西乡街道坪洲地铁B出口麻布新村七巷25号。
金属离子溶液配方(g/L):CoCl2·6H2O 0.2;CuCl2·2H2O 1.0;Na2·EDTA 5.0;H3BO3 3.0;MnCl2·H2O 2.0;NiCl·6H2O 1.0;ZnSO4·7H2O 4.0;NaMoO4·H2O 2.0。
3)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。
4)DCW测定:取10mL发酵液,5000g离心5min倒掉上清,去离子水洗涤菌体2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。
5)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。
实施例1
考察总碳氮比对裂殖壶菌油脂及各脂肪酸含量的影响。500mL挡板摇瓶,装液量100mL,转速180rpm,分别在裂殖壶菌培养基里以葡萄糖作为碳源,谷氨酸钠为主要氮源,设置总碳氮质量比分别为20:1,10:1,5:1,发酵至120h时收集菌体进行各成分分析。发酵过程参数变化及结果见表1,菌体在发酵的前48h生长迅速,菌体密度和干重迅速增长,发酵2d后,所有组别菌体干重和细胞密度均达到最大值。其中在总碳氮质量比为10:1条件下,细胞密度和细胞干重明显优于氮源过量组(5:1)和碳源过量组(20:1),发酵的第2d,分别达到最大值45.55和86.2g/L。在发酵终点,C:N质量比为10:1条件下,细胞干重达到60.10g/L,油脂产量为7.44g/L。奇数碳脂肪酸占总脂肪酸的比例为9.27%,OCFA油脂产量为0.684g/L。而OCFA最大产量在发酵3d后,油脂产量是7.92g/L,奇数碳脂肪酸占总脂肪酸比例为9.89%,OCFA油脂产量为0.783g/L。OCFA产量相对于氮源过量组(C:N=5:1)和碳源过量组(C:N=20:1)最高产量0.193g/L(第1天)和0.572g/L(第4天),分别提高了305.7%和36.9%。
实施例2
考察不同有机氮源和有机氮源比例对裂殖壶菌发酵产OCFA的影响。设置6组实验,有机氮N1与无机氮N2质量比设置为1:1;2:1;4:1;8:1和全有机氮源和全无机氮源组,摇瓶发酵,装液量100mL,发酵5天,每组两个重复,基于实施例1的结论,总碳氮质量比设置为10:1,其他条件同实施例1,结果见表2。有机氮源有利于菌体生长,菌体生长速度随有机氮源比例增加而增加。而无机氮源则有利于奇数碳脂肪酸积累,其中全无机氮源组在发酵的第3天,奇数碳脂肪酸占总脂肪酸的比例达到最高值15.75%,其中在N1:N2为8:1条件下,细胞干重和密度在发酵终点达到最大值42.98g/L和68.55,并且发酵终点时,油脂产量占干重百分比最高为17.59%。高于全有机氮源组(15.91%),这将有利于油脂的纯化以及节省成本。而奇数碳脂肪酸占总脂肪酸比例最高为10.29%,产量最高为0.54g/L,仅次于全有机氮源的13.64%和0.679g/L。
虽然全有机氮源组的OCFA产量高于碳氮质量比8:1组,但是全有机氮的成本高,由实施例2结果可知,无机氮源的添加可以提高油脂含量,提高碳源和氮源的利用率,又可以节省生产的成本,减少能源浪费。
实施例3
考察温度对裂殖壶菌产OCFA的影响,本实验设置三个温度,分别为23℃,28℃和33℃,5L发酵罐装液量3L,盐酸和氢氧化钠调控pH为7,转速600rpm,通气为3vvm,其它条件和摇瓶一致。发酵120h,每12h取一次样,用于发酵参数评估,结果见表3。由表3可知,23℃发酵组,菌体生长较快,在发酵终点,油脂产量和油脂含量占干重百分比均达到最大值,分别为18.78g/L和24.31%。发酵3天后,菌体密度和细胞干重分别达到较大值,分别为127.8和78.25g/L。同时奇数碳脂肪酸占总脂肪酸比例达到最大值28.51%,产量5.288g/L。相对于28℃发酵组(2.747g/L)和33℃发酵组(1.463g/L),发酵的最大奇数碳脂肪酸产量分别提高了92.5%和261.5%。其中变化最大的是C15:0,在发酵24h到48h之间,C15:0占总脂肪酸比例变化最大,从9.12%增加到20.24%,同时,C15:0脂肪酸产量在发酵3天后达到最高值4.887g/L。相对于28℃(2.323g/L)和33℃(1.087g/L)条件下的最大值分别提高了110.4%和349.8%。由此可见,23℃发酵有利于裂殖壶菌大量积累奇数碳脂肪酸,尤其是C15:0。
表2不同有机氮与无机氮比例发酵结果对比
实施例4
考察温度和培养基组分优化后,5L发酵罐分批补料培养裂殖壶菌高密度发酵产奇数碳脂肪酸(OCFA)的影响。本实验综合实施例1-3的结论,5L发酵罐,装液量3L,其他条件同实施例3,发酵5天后提取油脂并测定脂肪酸组成,结果见图1-3。如图所示,在整个发酵期间,细胞密度和干重变化趋势一致,均随发酵时间的延长而呈现先快速增长,后缓慢增长,最后趋于稳定。最大的细胞密度和细胞干重分别为120和80g/L。谷氨酸钠在36h消耗完全,葡萄糖消耗速率在前48h达到最大,后期通过流加补料维持葡萄糖浓度在40g/L左右。油脂产量在发酵96h后达到最高值24.95g/L,占干重百分比最高为33%。从脂肪酸组成上看,奇数碳脂肪酸占总脂肪酸的比例随时间延长而逐渐积累,在24-36h之间变化最大。总奇数碳脂肪酸最高占比在发酵84h达到32.19%,其中C15:0脂肪酸占到23.2%。其产量最高达到7.48g/L。根据图2的结果,采用优化后的温度和培养基成分发酵,生物量(干重)和优化前的培养相比有了近一倍的提高,奇数碳脂肪酸占总脂肪酸比例达到32.19%,含奇数碳脂肪酸油脂产量达到7.48g/L,高于现有文献报道的通过基因工程获得的最高值3.32g/L,提高了125%。
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 (5)
1.一种提高裂殖壶菌发酵生产油脂中奇数碳脂肪酸产量的方法,其特征在于,将裂殖壶菌(Schizochytrium sp.)接种在发酵培养基中,进行低温有氧发酵,控制培养基中的总碳源和氮源质量比为5:1~20:1,所述的培养基中有机氮 N1 与无机氮N2质量比为1:1~9:1,温度在23±2℃,发酵时间为60~120h;
所述发酵培养基的组分为,每1L培养基中,葡萄糖20-80g,氯化钾0.5-1.5g,酵母粉5-10g,七水合硫酸镁4-8g,磷酸二氢钾3-5g,无水硫酸钠20-40g,谷氨酸钠5-40g,硫酸铵1-6g,金属离子溶液2-3mL,消泡剂 0.0005-0.001g;
所述金属离子溶液配方为:CoCl2·6H2O 0.2-0.5g/L;CuCl2·2H2O 0.5-1.0 g/L;Na2·EDTA 2.0-10.0 g/L;H3BO3 2.0-5.0 g/L;MnCl2·H2O 1.0-5.0 g/L;NiCl·6H2O 0.5-1.0g/L;ZnSO4·7H2O 2.0-5.0 g/L;NaMoO4·H2O 1.0-3.0 g/L;
所述裂殖壶菌(Schizochytrium sp.)为裂殖壶菌(Schizochytrium sp.)ATCC PTA-9695;
所述奇数碳脂肪酸为C15:0和C17:0。
2.根据权利要求1所述的方法,其特征在于,所述的培养基中总碳源和氮源质量比为10:1。
3.根据权利要求1所述的方法,其特征在于,所述的培养基中有机氮 N1 与无机氮N2质量比为8:1。
4.根据权利要求1~3任一项所述的方法,其特征在于,所述发酵时间为84±12h。
5.根据权利要求1~3任一项所述的方法,其特征在于,所述发酵条件为:pH为7~8,通气为3vvm,转速600rpm。
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