CN1662654A - 包含多不饱和脂肪酸的微生物油的制备 - Google Patents
包含多不饱和脂肪酸的微生物油的制备 Download PDFInfo
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- CN1662654A CN1662654A CN038143836A CN03814383A CN1662654A CN 1662654 A CN1662654 A CN 1662654A CN 038143836 A CN038143836 A CN 038143836A CN 03814383 A CN03814383 A CN 03814383A CN 1662654 A CN1662654 A CN 1662654A
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Abstract
本发明提供了一种生产微生物油的方法,包含用两阶段发酵法培养微生物,其中,在发酵终点前的最后阶段,碳源:被微生物消耗的速率大于其被添加到培养基中的速率;添加速率为≤0.30M碳/kg培养基;或者其限制了微生物生长的速率。微生物因此只有有限的碳源,以致它们优先地代谢各种脂肪或油脂类,而不是花生四烯酸(ARA),故而增加了ARA在细胞中的比例。然后可以以己烷为溶剂,从微生物中回收微生物油,其中含有至少50%的ARA和至少90%的甘油三酯。
Description
技术领域
本发明涉及到一种生产PUFA的方法,所述PUFA可以是微生物油中的,该方法包含:采用两阶段法培养微生物,然后从该微生物中回收微生物油。本发明还涉及用该方法获得的新颖的(例如微生物的)油。在所述的油中,50%或者更多的油脂类(或PUFA,例如在所述的油里)是花生四烯酸(arachidonic acid,ARA)。这种油可能具有低于2.5或2.0的低过氧化值(peroxide value,POV)和/或低于1.0的低茴香胺值(anisidinevalue,AnV)。本发明还涉及包含本发明的微生物油或利用本发明的微生物油制造的食品和食物添加剂。
背景技术
多不饱和脂肪酸(polyunsaturated fatty acid),或PUFA,可以在自然界中发现。各种各样不同的PUFA产生自不同的单细胞生物(藻类、真菌等)。一种特别重要的PUFA是花生四烯酸(ARA),它是若干种长链多不饱和脂肪酸(LC-PUFAs)之一。化学上,花生四烯酸是顺式-5,8,11,14-二十碳四烯酸(20∶4),属于LC-PUFA的(n-6)族。
花生四烯酸是多种具有生物活性的化合物的主要前体,这些化合物合称为类二十烷酸(eicosanoid),其中包括前列腺素(prostaglandin)、凝血噁烷(thromboxane)和白细胞三烯(leukotriene)。花生四烯酸还是人类母乳脂类成分(lipid fraction)的组分之一,并被认为对婴儿的理想神经发育来说不可或缺。花生四烯酸具有各种各样的不同应用,包括用于婴儿配方食品(infant formula)、食品和动物饲料。
花生四烯酸可以利用微生物特别是利用丝状真菌Mortierella来生产。然而,花生四烯酸在得到的微生物油中的百分比通常非常低。人们为此进行过很多尝试,试图提高Mortierella的花生四烯酸产量,但是取得的成功却程度各异。很多提高花生四烯酸水平的尝试,都包括在工业环境中不易使用的步骤。
例如,Broshin et al,Process Biochemistry:(35)2000,pp1171-1175,将培养物在发酵结束后放置一周左右的时间。其中提到的ARA的量是基于生物量(biomass)(而不是基于从其中提取出来的油)的,因为该篇文献中没有描述过任何油的提取。Totani et al,Industrial Applications of singlecell oils,American Oil Chemists’Society Campaign,1992,Chapter 4,pp52-60和Lipids,Vol.22 No.12(1987),pages1060-1062,提出了用罕见的低发酵温度进行发酵,这意味着发酵会相当地慢。此处的ARA含量基于氯仿/甲醇溶剂混合物的萃取物。另一篇花生四烯酸制造领域的文献是WO96/21037。
EP-A-1035211(Suntory)描述了一种从M.alpina中生产ARA和双高-γ-亚麻酸(dihomo gamma-linolenic acid,DHGLA)油脂类的方法。然而,其ARA含量的计算基于生物量(而非从中提取的油)或由一种分析方法产生,其中多不饱和脂肪酸先被酯化,然后再用一种溶剂来萃取(而不是先行萃取,产生一种油,再在这种油的基础上测定出ARA的含量)。
一篇关于ARA的更高产量的报道宣称,采用M.alpina的1S-4株系,在收获的菌丝体中浓度接近70%(Shimizu S.,Oils-Fats-Lipids 1995,Proc.World Congr.Int.Soc.Fat Res.,21st(1996),Meeting Date 1995,Volume 1,pages103-109和Biochemical and Biophysical Research communications,Vol.150(1),1988,pages335-441)。但是,这个百分比是基于细胞的,与微生物油中的ARA的百分比并不一样。事实上,制成的油仅提供了39.0%的ARA含量(表27.2,第105页)。(注意,本领域中使用了各种不同的方式来测量ARA的含量,它们与本说明书后面所提供的数据不一定具有相同的单位或基于相同的分析方案)。此外,这还仅仅是在M.alpina细胞于发酵后再在室温中被多放置了六天才得到的,而对工业生产过程来说这明显不是可行的选择。
因此,我们需要找到能提高微生物油中花生四烯酸的比例(及产量)的方法,特别是工业规模上能用到的方法。
发明内容
本发明提供了多种新颖的方法,以生产含有的花生四烯酸比例提高的(微生物)油。这意味着花生四烯酸可以以更低的成本以及更高的效率地被生产出来。此外,由于本发明并不依靠对涉及到的微生物进行遗传修饰来提高花生四烯酸的产量,因此本发明有助于满足对天然的、未经遗传修饰的食物成分的日益增加的需求。而且这种油具有低氧化势(OxidationPotential),所以适合添加到毒性对之特别重要的人类食物中,例如婴儿配方食品。
因此,本发明的第一方面涉及到一种生产微生物油或多不饱和脂肪酸(PUFA)的方法。该方法包含:在发酵容器中以合适的培养基发酵(或培养)微生物,在发酵终点前(或在其之前的阶段);
a)碳源被微生物消耗的速率大于其被添加到培养基中的速率;
b)碳源以每小时≤0.30M碳/kg培养基的速率被添加;
c)碳源使得微生物生长的速率被限制,或对碳源进行控制,从而使得微生物代谢(它自己的)脂肪(类)和/或油脂(类);
d)添加碳源的速率被降低至碳源被微生物消耗的速度,或低于碳源被微生物消耗的速率;
e)在发酵终点或之前,碳源已经被全部用光,或其在培养基中的浓度接近于零;
f)碳源的添加被终止,但发酵可以继续;和/或g)对微生物所处的条件进行调节,使得它们优先于ARA进行脂肪(类)(举例来说,在细胞内,例如是PUFA)而不是ARA的代谢。
本发明的第二方面涉及到含有至少50%(或至少50.5%、51%或52%)的花生四烯酸(ARA)的微生物油。它也可能含有高达55%、57%或60%的ARA。这种油可以具有:
a)含量为至少90%的甘油三酯;
b)低于2.5的过氧化值;
c)低于1.0的茴香胺值;和/或
d)含量低于5%的磷脂。
上述的油可以通过第一方面所述的方法来制备。它可以是被己烷萃取的。
人们认为,一旦碳源浓度降低或受控制,细胞将开始代谢那些胞内的脂肪或油脂类。但是,这些细胞首先消耗脂肪而不是ARA。在这种情况下,ARA在细胞内脂肪或油脂类中的比例得以增加。因此,通过这种方式,第一方面所述的方法能够制得第二方面中所述的ARA含量更高的油。
具体实施方式
微生物
所用的微生物可以是细菌、酵母、藻类或真菌。优选使用真菌,特别是使用丝状真菌。优选的真菌是Mucorales目的。所述的真菌可以是Mortierella、Phycomyces、Entomophthora、Pythium、Thraustochytrium、Blakeslea、Rhizomucor或Aspergillus属的。优选的真菌是Mortierellaalpina种的。优选的酵母是Pichia或Saccharomyces属的,例如Pichiaciferrii。细菌可以是Propionibacterium属的。合适的藻类是甲藻(dinoflagellate)和/或属于Crypthecodinium、Porphyridium或Nitschia属的,例如是Crypthecodinium cohnii种的。
本发明所使用的微生物株系可以是自然界中存在的或是常用的工业株系。该株系可以是未经过遗传改造的,例如,它可以没有被载体转化过,或者不包含外源基因。由于目前在一些地区人们偏好那些不包含遗传工程成分的食物,因此所采用的微生物可以是未经修饰的株系。
多不饱和脂肪酸(PUFAs)
PUFA可以是一种PUFA,也可以是两种或更多种的不同的PUFA。
该PUFA或每个PUFA可以是n-3或n-6族的。优选的是C18、C20或C22的PUFA。它可以是至少具有18个碳原子和/或3或4个双键的PUFA。所述的PUFA可以被分离成游离脂肪酸、盐的形式,作为脂肪酸酯(例如甲酯或乙酯),作为磷脂和/或甘油单、双或三酯的形式。
适合的(n-3和n-6)PUFA包括:
二十二碳六烯酸(docosahexaenoic acid,DHA,22∶6Ω3),较为适合的是来自藻类或真菌,例如(甲藻)Crypthecodinium或(真菌)Thraustochytrium;
γ-亚麻酸(γ-linolenic acid,GLA,18∶3Ω6);
α-亚麻酸(α-linolenic acid,ALA,18∶3Ω3);
共轭亚油酸(conjugated linoleic acid,十八碳二烯酸(octadecadienoicacid),CLA);
双高-γ-亚麻酸(dihomo-γ-linolenic acid,DGLA,20∶3Ω6);
花生四烯酸(ARA,20∶4Ω6);和
二十碳五烯酸(eicosapentaenoic acid,EPA,20∶5Ω3)。
优选的多不饱和脂肪酸包括花生四烯酸(ARA),二十二碳六烯酸(DHA),二十碳五烯酸(EPA)和/或γ-亚麻酸(GLA)。具体而言,优选为花生四烯酸。
发酵
典型的发酵/培养是在含有(液体,通常为水性的)培养基的合适的发酵罐(或发酵容器)中进行的。一般由小的进料发酵罐(feed fermenter)向主发酵容器进行无菌接种。典型采用的为深层(submerged)和/或需氧的发酵过程。这可以在深池(deep tank)发酵罐中进行。发酵罐可以配备用以监控和/或改变pH和温度的设施。此外,这种容器还可以适于进行、或能够实现通气和/或细胞与液体的混合,比如,溶液的搅拌。上述操作可以是搅动(stirring),例如通过机械手段获得的。
发酵罐适合的体积为至少10、20、40或者甚至60立方米。高达100或者甚至150立方米的体积也是可以使用的。
典型的发酵将持续10天或更短,优选9天或更短,更优选的是8天或更短。它可以是至少4、5、6或7天。
可供选择地,发酵可以为150至200小时,例如160至190小时,举例来说,170至180小时。发酵终点通常是搅拌振荡和/或通气停止的时刻。这可以是发酵容器和/或辅助设备被(有效地)关掉之时。然后微生物可以从发酵罐中被移走。
发酵可以在20℃至40℃之间的温度下进行。
碳源和氮源
所有合适的培养基都可以用于发酵,例如,一种适用于所使用的微生物的培养基。碳源可以包含(复杂的来源,例如)麦芽糖糊精、燕麦粉、燕麦片、糖蜜、植物(例如大豆)油、麦芽提取物、淀粉、乙醇或大豆油。优选的(不复杂的)碳源包括碳水化合物或糖,例如果糖、麦芽糖、蔗糖、木糖、甘露醇、葡萄糖或乳糖或甘油(glycerine)(例如,来自植物来源的)、柠檬酸、醋酸、丙三醇(glycerol)、乙醇或抗坏血酸(例如其钠盐)。在本发明的一种优选实施方式中,碳源是葡萄糖或者包含葡萄糖的,特别地,是葡萄糖浆。
适合的氮源包括酵母提取物、尿素以及蛋白胨。培养基可以不含琼脂。
优选的氮源和/或碳源是水溶性的或者是易与水混合的。
培养基中的个别成分(例如氮源和/或碳源)可以于发酵起点就(全部)存在,可以在发酵期间连续不断地添加,也可以分批或逐步地添加。具体而言,培养基中存在的碳源的量,将会典型地按照如下所述的方式被控制,优选是依靠控制添加碳源的速率。
氮和/或碳源可以被单独补充(或添加),也可以被同时补充,还可以以整合在一起的制剂的形式(combined preparation)被补充。它们可以存在于同一组合物中(如果被认为是必需的话),该组合物优选是一种液体。碳源和/或氮源(向发酵罐容器中)的添加,可以在真菌细胞被加入(到容器中)之前,换句话说是在接种之前进行,也可以是在发酵期间;或者它们可以在发酵之前以及发酵期间被添加。
培养基
培养基优选为一种水性液体。它还可以另外包括其它物质在发酵中进行辅助,例如,螯合试剂(比如柠檬酸)、消泡剂(比如大豆油)、维生素(比如硫胺素(thiamine)和/或核黄素(riboflavin))、任何必需的催化金属(比如,镁或钙等碱土金属,或者锌或铁和/或其它金属诸如钴和铜)、磷(比如磷酸盐)和/或硫(比如硫酸盐)。如果必要的话,该培养基可以包括一种附加的油,例如,橄榄或者大豆油,但是,优选地,培养基不包括上述的油。
(最适的)生长(或发酵)温度可以根据所使用的微生物而变化。但是,优选是20℃至40℃,更优选的是22℃至30℃或32℃。特别地,进行发酵的温度是在≥22℃或者≤25℃,诸如22℃至30℃,举例来说是23℃-28℃。发酵期间水性液体的pH可以是4至10,例如5至8,最适为6至7。
发酵期间,典型做法是对培养基进行搅动或搅拌振荡来帮助实现通气。水性液体和细胞被适当地混合或搅拌。这可以通过进行通气来实现,例如将一种气体,如空气,向水性液体中鼓泡。这还可以达到一个附加的目的,即向真菌细胞提供氧气:因此发酵优选为需氧发酵。搅拌或混合的其它手段包括搅动,例如使用叶轮。它可以是水翼轴流式(hydrofoil axialflow)设计,也可以被设计成使水性培养基从叶轮(例如涡轮)向外呈放射状被推动的形式。即使没有搅动,发酵过程中向微生物细胞提供氧气也是优选的,所以,在此处,通气(例如,通过鼓入空气、氧气或其它包含氧气的气体)是有益的。通气可以是0.1至2.0vvm,例如从0.5至1.0vvm。
优选的发酵罐体积为至少2升或5升,优选为至少10升。然而,对于工业上使用的或达到工业规模的发酵罐来说,容器体积优选为至少50、100、500或者1000升。
发酵的最后(或第二)阶段
发酵过程可以被分为至少两个阶段。第二或最后阶段,可能马上就要到达发酵终点,其特征为微生物可用碳源的量降低或是第一方面中给出的(a)到(g)特征中的任意一项。典型地,该阶段起始于发酵终点前15至2个小时,优选是发酵终点前10小时或更短,更加优选的是发酵终点前的3至5小时。优选地,该阶段典型地起始于发酵开始之后10天以内,更加优选的是起始于发酵开始之后的9天以内,进一步优选的是发酵开始之后的8天以内。
在发酵的第一或是初期阶段,碳源可能是过量的。因此可利用的碳源的量无法限制微生物的生长。添加碳源的速率可能会超过其被微生物消耗的速率。在发酵的第二或是最后阶段,添加进去的碳源的量可以被减少或完全停止。这意味着,在发酵的第二或是最后阶段,可为微生物所用的碳源的量将会减少。典型地来说,在第二,最终或是最后阶段,或者接近发酵终点之时,碳源可能:
被微生物消耗的速率大于其被添加到培养基中的速率(例如添加速率低于消耗速率);
以每小时≤0.30M碳/kg培养基的速率被添加,例如≤0.25或≤0.20M碳/kg培养基/hr,但是至少为0.01、0.02或0.05M碳/kg培养基/hr(此处的单位表示碳源中碳的摩尔数或摩尔量,而非碳源本身的重量或摩尔数);
限制了微生物的生长和/或(多不饱和脂肪酸的)生产的速率。
在第二阶段,典型的碳源浓度为≤10g碳源/kg培养基,优选为0.01g/kg或0.1g/kg至8g/kg或10g/kg,更优选的是0.5g/kg至5g/kg,进一步优选为1g/kg或2g/kg至4g/kg或5g/kg。这意味着,在发酵的最后阶段,平均起来,每kg培养基中会有≤0.30M的碳,优选是每kg为0.003M至0.3M的碳。每kg0.015M至0.17M的碳是有益的,而进一步优选的是每kg0.03M至0.17M的碳。
如果碳源包含葡萄糖,(在最后阶段)葡萄糖的典型浓度平均为每kg培养基中≤10g,优选是0.01g/kg或0.1g/kg至8g/kg或10g/kg。0.5g/kg至5g/kg是有益的,进一步优选为1g/kg或2g/kg至4g/kg或5g/kg培养基。在此种情况下,培养基中包含细胞及水性培养基,换句话说,就是“培养液”(细胞和周围的液体)。
在最后阶段碳源添加速率优选为不超过每kg0.03M碳,优选的是不超过0.025或0.02M碳/kg(培养基)。优选的添加速率在0.015M碳/kg左右。如果碳源是葡萄糖,那么葡萄糖添加速率优选为每小时小于1.0g/kg培养基,例如小于0.8g/kg培养基,例如小于0.5g葡萄糖/kg培养基。
最后阶段中碳源添加速率优选为碳源被微生物消耗的速率的一半左右。但是,添加速率∶消耗速率这个比率可以在1∶1-3中变化,例如从1∶1.5至2.5,最适的为从1∶1.8至2.2。或者,添加速度可以是消耗速度的30%-70%,例如40%至60%,最适为45%至55%。
在发酵的第二阶段,适当的碳源浓度可以通过仔细地控制碳源的添加速率来获得。典型地,它将会在最后阶段中适当地降低,或在最后阶段的一开始就陡然下降。对培养物的定期采样和分析被用来测定碳源的浓度,并对碳源的添加速率做出必要的调节。这可以通过使用一套计算机系统来自动完成。
巴氏消毒(Pasteurisation)过程
巴氏消毒通常发生于发酵完成之后。在一种优选实施方式中,巴氏消毒将终止发酵,因为巴氏消毒期间的热度会杀死细胞。巴氏消毒因此可以针对发酵培养液(或液体(水性)培养基中的细胞)进行,虽然其也能够针对从培养液中获得的微生物生物物质(biomass)进行。在前一种情况中,巴氏消毒可以发生于微生物细胞仍处于发酵罐中时。巴氏消毒优选于微生物细胞受到更多的处理前进行,例如微粒化(比如通过挤压)、粉碎或者捏炼(kneading)。
优选地,巴氏消毒方案足以抑制或失活一种或多种能使多不饱和脂肪酸或微生物油降解或对其产生不良影响的酶,例如脂肪酶。
一旦发酵已被终止,就可以过滤发酵培养液,或者进行其它处理以去除其中的水或水性液体。水分被移除之后,可以获得一种生物物质“压滤渣(cake)”。如果巴氏消毒还未进行,就会对这些去水的细胞(或生物物质压滤渣)进行巴氏消毒。
油的提取
如果需要的话,例如,在发酵被终止之后,微生物可以被杀死或对其进行巴氏消毒。这可以用来失活任何不受欢迎的酶,例如那些可能使上述的油降解或降低PUFA产量的酶。
在培养或发酵完成或已经结束之后,发酵培养液(细胞和水性液体)就可以从发酵罐中被移出来,如果必要的话,可以将液体(通常是水)从其中移走。任何适合的固液分离技术都是可以使用的。其(去水)可以依靠离心和/或过滤。细胞可以被漂洗,比如说采用水溶液(例如水),达到诸如去除细胞外水溶性或易分散于水中的化合物的目的。然后可以从微生物中回收得到油,例如使用溶剂使这种油得以被溶剂萃取,优选是己烷萃取。
这种油可能不含有(或基本去除了)GLA和/或DGLA。
PUFA的提取过程
所述的PUFA(或通常含有该PUFA的微生物油)可以自包含细胞的(比如,·干燥的)微粒(例如,挤压得到的)中提取。这种提取可以使用溶剂来进行。优选使用无极性溶剂,例如C1-8,比如C2-6,链烃,举例来说是己烷。也可以使用二氧化碳(液态形式的,比如处于超临界状态的)。
然后可以对上述细胞进行提取,例如使用有机溶剂,优选是在氮流中进行的。其它可用的有机溶剂包括乙醚、甲醇、乙醇、氯仿、二氯甲烷和/或石油醚。使用甲醇及石油醚和/或使用由氯仿、甲醇和水构成的单层溶剂体系来萃取也是可行的。有机溶剂从萃取物中减压蒸发,可以得到含有高浓度花生四烯酸的微生物油。
优选地,溶剂被允许从干燥微粒中滤出。适合的微生物微粒化和挤压技术以及后续的含有PUFA的微生物油的提取,在WO-A-97/37032中进行了描述。
上述溶剂可以令我们获得一种含有PUFA的粗制的(crude)油。这种油在此状态即可使用,无需更多处理,它也可以再经过一步或数步精炼。PCT/EP01/08902号国际专利申请(该文件的内容和本文中所述及的其它所有文件的内容通过引用的方式被包括在本文中)中描述了合适的精炼方案。例如,可以对所述的油进行酸处理或脱胶(degumming)、碱处理或去除游离脂肪酸、脱色(bleaching)或去除色素、过滤、冬化(winterisation)(或冷却,例如为了去除饱和的甘油三酯)、脱臭(或对游离脂肪酸的去除)和/或上光(polishing)(或对油不溶性物质的去除)。
由此得到的油特别适合营养上的用途,可以被添加到(人类的)食物或(动物的)饲料中。例子包括奶、婴儿配方食品、健康饮品、面包和动物饲料。
提纯/精炼
微生物油可以被精炼和提纯。该过程可能涉及到去除下述组分中的一种或数种:磷脂、痕量金属、色素、碳水化合物、蛋白质、游离脂肪酸(free fatty acid,FFA)、油溶性物质、水溶性物质、皂(soap)或皂化(saponified)物质、氧化产物、硫、甘油单或双酯、色素分解产物、溶剂和/或固醇。提纯可以减少或去除“异味(off-flavours)”和/或增加油的稳定性。
为达到该目的,上述方法(例如提纯)可以包括脱胶(或酸处理)、中和(或碱处理)、水洗、脱色、过滤、脱臭、上光和/或冷却(或冬化)。提纯优选为包括酸处理和/或碱处理(脱胶和中和)。或者,纯化方法可以包括脱色和/或脱臭。然而提纯将优选包括脱色和/或脱臭,另外,最优地,还要加上酸和/或碱处理。
油
本发明的第二方面提供了微生物油,它包含至少35%或40%的至少一种PUFA,例如ARA。上述的油可以具有至少50%、55%或60%或更多的这种PUFA,例如ARA。它可以具有至少90%的甘油三酯。这种微生物油优选包含50%、55%或60%至90%的花生四烯酸,更优选的是60%至80%,进一步优选的是60%至70%的花生四烯酸。
上述微生物油优选含有90%至100%的甘油三酯,例如至少90%或96%,优选为至少98%,更优选的为至少99%,而最优的超过99.5%。典型地,这种油中二十碳五烯酸(EPA)的含量低于5%,优选为低于1%,而更优选的是低于0.5%。该油中,C20、C20∶3、C22∶0和/或C24∶0多不饱和脂肪酸(PUFAs)中任何一种的含量可以少于5%,少于2%,少于1%。游离脂肪酸(FFA)的含量可以≤0.4%、0.2%或0.1%。
甘油三酯中存在的PUFA中,优选至少40%,例如至少50%,而更优选为至少60%,都处于甘油的α位(出现于甘油三酯的主链(backbone))也就是所谓的1或3号位。上述PUFA中处于β(2)位的优选为至少20%,例如至少30%,更优选的为至少40%。
上述油中的磷脂含量最大值适合为5%、3%或者2%和/或最小值可以是0.1%、0.5%或者1.0%。
典型地,上述微生物油可以通过本发明第一方面中的方法得到。上述的油优选从真菌中被分离出来,更优选地,上述的油是从Mortierella特别是M.alpina中被分离出来的。上述的油被己烷适当地萃取。
ARA的含量
纯粹为了清楚起见,ARA的百分含量的计算将被解释一下,尤其是因为文献中可能有时在不同的基础上计算ARA的含量。所述ARA的百分比基于上述的油(即已经从生物物质中被提取出来的那些),而不是生物物质本身。其以重量/重量为基础。该含量基于由己烷萃取出来的油,所以是基于己烷可萃取的油脂类(hexane extractable lipids,HEL)的。其基于油的总量,而不是基于脂肪酸的总量(很多时候这会给出一个令人误解的更高数值)。ARA的含量通过公知的FAME分析方案(使用脂肪酸甲酯)测定,细节参见AOCS Ce1 b89。不同的溶剂萃取不同的油脂。注意,在这种情况中,上述的油是先经己烷萃取的,然后再通过FAME分析来测定ARA的含量。这和先行酯化花生四烯酸(例如还在细胞中的时候)然后再提取得到的甲酯做进一步分析所给出的结果将会是不一样的。
过氧化值(POV)
上述微生物油的POV优选为不超过3.0、2.5或2.0。但是,使用本发明的方法,还可以获得低得多的POV,这些数值可以低于1.5或低于1.0。低于0.8和甚至低于0.4的数值也能够被得到。
茴香胺值(AnV)
上述微生物油的茴香胺值优选为不超过1.0,例如不超过0.6、0.3或者甚至不超过0.1。
用途和产品
本发明的第三方面涉及到一种组合物,其中包含第二方面中所述的油,以及如果合适的话,其中还具有一种或多种(额外的)物质。该组合物可以是为动物或人类提供的食品和/或食物添加剂。在本发明的供人类消费的多种实施方式中,上述的油可以经过加工,使其适合人类消费,典型的是对从微生物中获得的油进行精炼或提纯。
上述组合物可以是婴儿配方食品或(人类)食品。此处的配方食品的成分可以经过调节,使其含有的各种油脂或多不饱和脂肪酸的量与正常的母乳相似。上述调节可以包括将本发明的微生物油与其它油类混合,以获得适当的组合物。
上述的组合物可以是一种动物或海产饲料组合物或添加剂。此类饲料和添加剂可以供给任何的养殖动物,特别是绵羊、牛和家禽。此外,上述饲料或添加剂还可以供给被养殖的海洋生物,例如鱼和有壳水生动物。上述组合物因此还可以包含一种或多种适于上述动物的饲料物质或成分。
本发明的油可以作为油直接售卖,也可以被包纳于合适的包装中,典型的是一种内部涂上环氧酚紫胶漆(epoxy phenolic lacquer)并且经过充氮(flushed with nitrogen)的铝瓶。上述的油可能含有一种或多种抗氧化剂(例如,生育酚,维生素E,棕榈酸(palmitate)),每种的浓度例如为50至800ppm,举例来说是100至700ppm。
适当的组合物可以包括药用或动物治疗用的(veterinary)组合物,例如,口服或化妆品用途的组合物。这种油可以按照上述方式被使用,或者还可以被装入胶囊,例如在一种壳(shell)里,因此可以成为胶囊的形式。这种壳或胶囊可以包含明胶和/或甘油。上述组合物可以包含其它成分,例如调味料(比如柠檬或酸橙(lime)香料)或一种药用的或动物治疗可接受的载体或赋形剂。
本发明的一个方面的优选特征和特性在进行必要的修正后同样适用于另一个方面。
下述的实施例只是被用来阐述本发明的,而非用来对本发明进行限制。
对照实施例1和2以及实施例3和4
花生四烯酸的生产
装有Mortierella alpina菌株CBS 168.95(由DSM N.V.,2600MA Delft,荷兰(该机构已授权本申请人使用该被保藏的生物材料)于1995年2月20日提交至Centraal Bureau voor Schimmelcultures(CBS),P.O.Box 85 167,3508 AD Utrecht,荷兰进行保藏,保藏编号为DS 30340)的1ml小瓶被贮藏于-80℃,并在无菌条件下打开。其内容物被用来接种到含有100ml培养基的500ml摇瓶(flask)里,所述的培养基含有(g/l):
葡萄糖,20;
酵母提取物(Gistex糊状物(固体80%,蛋白质(Nx 6.25)46%,NaCl 16%,pH(2%溶液)5.6,灰分22%,细菌总数(total platecount)104/g,肠内细菌(Enterobacteriaceae)<10/g,大肠杆菌<1/g,酵母菌及霉菌<100/g,来自DSM N.V.,Savory Ingredients POBox 1,2600MA Delft)),12.5;消泡剂(Basildon 86/013K硅/无硅消泡化合物,参照厂商说明书使用,Basildon Chemical Company,Kimber Road,Abingdon,Oxford,England OX14 IRZ),0.2。
上述培养基的pH在高压灭菌前被调至7.0。
培养物在25℃以250rpm的摇速培育48小时,然后用于向四只含有500ml培养基的2000ml摇瓶中接种,所述的培养基包括(g/l),葡萄糖,20;酵母提取物(Gistex糊状物),25;消泡剂(Basildon86/013K),0.2。灭菌前的pH为7.0。
上述培养物在25℃被培育24小时,然后被用来移种(seeding)至一只5m3的接种发酵罐(inoculaion fermentor)中,罐中有2400升与上述2000ml瓶中所用的成分相同的培养基(灭菌前的pH为6.0)。
发酵温度被设置在25℃,振荡速度为150rpm,容器压力是0.5bar,通气率为0.5VVM。
接种体发酵罐(inoculum fermenter)中的培养物在大约36小时后(氧气吸收速率>3mmol/kg/h)被转移至主发酵罐中。
主发酵罐含有(g/l):
葡萄糖,35;
酵母提取物(Expresa 2200粉,低钠啤酒酵母,蛋白胨(提取物),固体>96%,总氮量>10%,氨基氮6-7%,NaCl<1%,pH(2%溶液)5.3-6.3,灰分<12.5%,均匀粉末,来源为DSM N.V.,Savory Ingredients),5.0;
NaH2PO4·2H2O,1.0;
KH2PO4,2.0;
MgSO4·7H2O,0.5;
Basildon 86/013K,0.3;柠檬酸·1H2O,0.6;
ZnCl2,0.010;
Fe2(SO4)3 20%H2O,0.025;
MnSO4·1H2O,0.010;(灭菌前的pH5.0)。
葡萄糖单独灭菌,在灭菌后加入到主发酵罐中。
发酵持续175小时。培养基的pH持续在pH6(+/-0.1)左右,伴随0.5VVM的通气(空气流),空气压力为0.8bar,以70rpm振荡。通过连续增加振荡速率至100rpm及连续增加空气流至0.9VVM,使氧气水平保持在D.O.≥30%。
已灭菌的50%(质量/质量)左右的葡萄糖溶液被添加至发酵罐中,以使葡萄糖浓度保持在10g/l以上,另外,从大约30至78小时,625kg的25%的酵母提取物溶液也被进料至发酵罐,添加速率被加以控制使得氨浓度<30mg/l。
上述实验被进行了四次(实施例1至4),培养基中的葡萄糖浓度被随时间监控。图1显示了以g/kg表示的葡萄糖浓度相对于进入发酵的小时数的图表。其中显示,实施例4的最后一个数值为在172小时,2.2g/kg。这是发酵终点(end of fermentation,EoF)前3小时。葡萄糖水平在发酵终点前一小时左右为零。
在对照实施例1和2中,发酵终点时,碳源(葡萄糖)的浓度大大超过5g/kg。事实上,在发酵终点前10小时,葡萄糖浓度在20g/kg左右。因此,在实施例1和2中,葡萄糖浓度不会对微生物群或对ARA的生产加以限制的水平。
在实施例3和4中,发酵的最后阶段即将达到发酵终点时,葡萄糖的浓度被控制,使得在发酵终点前约10小时,葡萄糖浓度为5g/kg左右。在该最后阶段,超过10小时,葡萄糖以每小时0.5g/kg的速度被添加。在发酵终点葡萄糖浓度实质上为零。在此阶段,葡萄糖的消耗速率大约是添加速率的两倍,即约1g/kg/hr。
表1至4(与实施例1至4对应)显示了发酵过程中,培养基内的葡萄糖浓度(g/kg)随时间的变化。
表1
时间(小时) | 葡萄糖浓度g/kg |
0 | 48.7 |
24 | 57.6 |
28 | 47.8 |
54 | 46.4 |
78 | 62.0 |
102 | 62.5 |
126 | 48.2 |
150 | 42.5 |
167 | 20 |
表2
时间(小时) | 葡萄糖浓度g/kg |
0 | 47.8 |
28 | 32.5 |
54 | 32.2 |
78 | 41.3 |
102 | 49.5 |
126 | 46.8 |
150 | 29.9 |
165 | 17.1 |
表3
时间(小时) | 葡萄糖浓度g/kg |
0 | 49.2 |
28 | 60.1 |
54 | 40.8 |
78 | 34.0 |
102 | 37.3 |
126 | 23.2 |
150 | 16.7 |
172 | 7 |
表4
时间(小时) | 葡萄糖浓度g/kg |
0 | 45 |
28 | 34.1 |
54 | 34.7 |
78 | 29.2 |
102 | 38.1 |
126 | 45 |
150 | 23.5 |
172 | 2.2 |
在发酵终点,微生物和周围的水性液体(发酵培养液)都从发酵罐中被移出。培养液经过固液分离来去除一些水分。然后,剩下的细胞被挤压,再用己烷进行溶剂萃取。从而,由分别经历了上述四个不同的发酵方案的细胞中,获得了含有微生物油(己烷可萃取的油脂类)的花生四烯酸。
使用公知的FAME分析方案(细节在AOCS Ce1b89中)来测定ARA在油中的百分含量(基于质量/质量基础的)。在实施例3中,微生物油中ARA的浓度为508g/kg(50.8%)。例4中相应的数据为545g/kg(54.5%的ARA)。作为比较,在对照例1和2中,从细胞中提取出来的微生物油要少得多,分别为36.8%和36.7%。
荷兰真菌保藏中心
真菌生物多样性中心
荷兰皇家科学院研究所(KNAW)
DSM N.V.
Postbus 1
2600 MA DELFT
Nederland
乌德勒支(Utrecht),2003年5月12日
我方编号:0183/12mei2003
安全保藏声明
荷兰真菌保藏中心(CBS),乌德勒支,荷兰特此声明,下述微生物:
CBS 168.95 Mortierella alpina DS30340
于1995年2月20日被保藏至荷兰真菌保藏中心,其将作为CBS收到的安全保藏物被保密地贮藏,在最近一次交纳年度维持费后至少一年的时间内,其会被妥善地保持存活及不受污染。
在作为安全保藏物被贮藏的期间,所述的微生物将只提供给保藏者或经由保藏者授权的人。
只要保藏者通知CBS被保藏的菌株不应再被继续维持,在收到保藏者请求后,CBS就将毁灭所述的株系。CBS需在当年度结束前收到上述请求。
CBS专利管理部
J.A.Stalpers博士
(馆长)
被保藏的生物材料仅可被专家获得的要求
“对上述行为可行的所有指定国而言,在上述行为可行的范围以内,并在该指定国法律所允许的范围以内,根据相关的专利法规,例如欧洲专利公约细则28(4)、英国专利条例1995第2部分第3段、澳大利亚规则3.25(3)以及其它任何指定国的略有差别但基本类似的规定,要求所述被保藏的生物材料的样品仅可由独立专家获得。”
Claims (11)
1.一种生产多不饱和脂肪酸的方法,所述的方法包含:在发酵容器里的培养基中培养微生物,由此在发酵终点之前的阶段:
a) 碳源被微生物消耗的速率大于其被添加到培养基中的速率;
b) 碳源以每小时≤0.30M碳/kg培养基的速率被添加;
c) 碳源使得微生物生长的速率被限制,或对碳源进行控制,从而使得微生物代谢脂肪(类)和/或油脂(类);
d) 添加碳源的速率被降低至碳源被微生物消耗的速率,或低于碳源被微生物消耗的速率;或
e) 在发酵终点或之前,碳源已经被全部用光,或其在培养基中的浓度接近于零;
f) 碳源的添加被终止,但发酵可以继续;和/或
g) 对微生物所处的条件进行调节,令其优先于花生四烯酸(ARA),代谢或消耗一种或多种脂肪(类)或油脂(类)。
2.如权利要求1所述的方法,其中:
h) 在所述阶段,碳源的浓度平均≤10g/kg和/或≤0.17M碳/kg培养基;
i) 所述的碳源是葡萄糖;和/或
j) 所述的多不饱和脂肪酸出现于微生物油中。
3.如权利要求1或2所述的方法,其中:
k) 第二阶段起始于发酵终点前15至2小时,或在发酵起点后的10天之内。
4.如前述权利要求中任意一项所述的方法,其中:
l) 所述的(整个)发酵进行的温度为≥22℃和/或≤30℃;
m) 是在没有附加的油的情况下进行的;和/或
n) 所述的发酵过程不长于9天。
5.如前述权利要求中任意一项所述的方法,其中:
n) 所述的多不饱和脂肪酸包括花生四烯酸(ARA);和/或
o) 所述的容器具有至少10升的容量。
6.如前述权利要求中任意一项所述的方法,其中所说的微生物是Mortierella,可以是Mortierella alpina,和/或是未经过遗传修饰的。
7.一种微生物油,包含至少50%的花生四烯酸(ARA),和:
a) 至少含有90%的甘油三酯;
b) 过氧化值(POV)不高于2.5;
c) 茴香胺值(AnV)不高于1.0;
d) 是己烷萃取的;和/或
e) 磷脂含量低于5%。
8.如权利要求7所述的油,包含:
f) 少于5%的C20和/或C24多不饱和脂肪酸(PUFAs);和/或
g) 少于5%的C22+多不饱和脂肪酸。
9.如权利要求7或8所述的油,其中:
h) 所述游离脂肪酸的含量≤0.4%;
i) 所述甘油三酯的含量为至少95%或98%,和/或
j) 所述的油可以通过如权利要求1至6中任意一项所述的方法进行制备。
10.一种组合物,包括可以通过如权利要求1至6中任意一项所述的方法获得的微生物油,和/或如权利要求7至9中任意一项所述的微生物油。
11.如权利要求10所述的组合物,所述组合物是食品(例如婴儿配方食品)、食物、饲料或饲料添加剂,药用的、动物治疗用的或化妆品用的组合物。
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EP (1) | EP2333093B1 (zh) |
JP (5) | JP2005530519A (zh) |
KR (4) | KR20140023448A (zh) |
CN (3) | CN101787379A (zh) |
AU (1) | AU2003281542A1 (zh) |
BR (2) | BR0311916A (zh) |
CA (1) | CA2489982C (zh) |
DK (1) | DK1513941T3 (zh) |
EA (2) | EA200901291A1 (zh) |
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CN105360351A (zh) * | 2010-01-19 | 2016-03-02 | 帝斯曼知识产权资产管理有限公司 | 生产二十碳五烯酸的微生物、脂肪酸组成物及其制作方法与用途 |
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CN105360351A (zh) * | 2010-01-19 | 2016-03-02 | 帝斯曼知识产权资产管理有限公司 | 生产二十碳五烯酸的微生物、脂肪酸组成物及其制作方法与用途 |
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CN103131529A (zh) * | 2011-11-23 | 2013-06-05 | 丰益(上海)生物技术研发中心有限公司 | 一种提取微生物油脂的方法 |
CN108902364A (zh) * | 2018-08-04 | 2018-11-30 | 望江县振兴植物油厂(普通合伙) | 一种儿童专用调和油及其制备方法 |
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