CN101560529B - 花生四烯酸及其生产和使用方法 - Google Patents
花生四烯酸及其生产和使用方法 Download PDFInfo
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- CN101560529B CN101560529B CN200910006766.XA CN200910006766A CN101560529B CN 101560529 B CN101560529 B CN 101560529B CN 200910006766 A CN200910006766 A CN 200910006766A CN 101560529 B CN101560529 B CN 101560529B
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Abstract
本发明涉及含花生四烯酸油脂的生产方法,其中基本上不含二十碳五烯酸。本发明还涉及含油组合物,油中含有高含量的甘油三酯形式的花生四烯酸,以及这类油脂的使用方法。在一种较好的实施方式中,在适宜条件下培养高山被孢霉(Mortierella alpina),该条件下能够生产出花生四烯酸残基含量特别高的甘油三酯,然后收获生物质,油脂经萃取、回收后用作婴儿配方食物中的添加剂。
Description
本申请是中国专利申请CN96192002.5(PCT/US96/00182),“花生四烯酸及其生产和使用方法”的分案申请。
发明领域
本发明涉及花生四烯酸的生产,含有花生四烯酸的组合物及其用途。
发明背景
花生四烯酸(ARA)是ω6类长链聚不饱和脂肪酸(PUFA)(5,8,11,14-二十碳四烯酸,20∶4)。ARA是人体中含量最丰富的C20PUFA。它主要存在于器官、肌肉和血液组织中,在血液、肝脏、肌肉和其它器官系统中作为主要与磷脂结合的结构脂类起着重要作用。除了主要的结构脂类作用之外,ARA还是许多循环二十烯酸衍生的生物活性物质(eicosenoid),例如前列腺素E2(PGE2),前列腺环素(PGI2),血栓烷A2(TxA2),和白细胞三烯leukotireneB4(LTB4)和C4(LTC4)的直接前体。这些eicosenoid具有对脂蛋白代谢,血液流变学,血管弹性,白细胞功能和血小板激活的调节作用。
尽管ARA对人体很重要,但是它不能在体内从头合成。ARA是通过必需脂肪酸亚油酸(LOA)的延长和去饱和来合成的。该过程需要酶Δ6-去饱和酶的存在,该酶在人体内含量很低,Burre等,脂类(Lipids),25:354-356(1990)。所以,大多数ARA必须靠饮食提供,这一点在身体的快速成长期,例如婴儿期,尤为重要。
婴儿在第一年中其体重可增至2至3倍。所以,需要大量的饮食ARA。为了满足这一需要的增长,母乳中含有大量的ARA。Sanders等,美国临床营养学杂志(Am.J.Clin.Nutr.),31:805-813(1978)。ARA是母乳中主要的C20PUFA。那些母乳喂养婴儿的母亲,尤其是素食主义者其婴儿可以通过附加的饮食ARA获益。但是,许多母亲不进行母乳喂养,或者不在整个婴儿快速成长期进行母乳喂养,而选择使用婴儿配方食物。
在本申请人所知的市售婴儿配方食物中都不含甘油三酯形式的ARA。再次引用参考的US4,670,285(Clandinin等)揭示了婴儿对包括ARA在内的脂肪酸的需求。为了提供这些脂肪酸,Clandinin等提出了一种蛋黄、鱼油或血红细胞磷酸酯和植物油的混合物,以此作为一种建议婴儿配方食物。但是,鱼油中含有大量的二十碳五烯酸(EPA)。已知EPA在婴儿体内抑制ARA的合成。Carlson等,INFORM,1:306(1990)。所以,最好能够提供ARA但不提供EPA。此外,蛋黄中的ARA含量较低,所以,Clandinin等的混合物在经济上并不可行。
由于ARA存在于动物油而不是植物油中,其工业量的生产仍然是一个希望但困难的目标。Shinmen等在微生物技术(Microbiol.Biotech.)31:11-16(1989)中报道,利用常规的搅拌罐发酵,由一种分离出的真菌高山被孢霉(Mortierella alpina)生产ARA。(参见Shinmen等的日本专利1,215,245)。在培养之后,收获微生物,干燥后用有机溶剂从真菌生物质中萃取脂类,然后对脂类进行化学(共价)改性。例如,水解脂类混合物或将其转化为醚酯,然后与环糊精混合,再用作食物补充。Shinmen等并没有说明或建议使用未经改性的微生物脂类。
Porphyridium cruentum,一种红色的微藻,可以在池塘中大量种植,而且其脂含量中含有多至40%的ARA。Ahern等,生物技术与生物工程(Biotech.Bioeng)25:1057-1070(1983)。不巧的是,这些ARA主要与半乳糖脂结合,这是一种母乳中没有的复合极性脂。这样,不仅生成的可利用ARA仅占生物量的1%,而且ARA的形式如果不经改性不适合用作婴儿配方食物的添加剂。
US4,870,011(Suzuki等)公开了从被孢霉属(Mortierella属)真菌中获得例如γ-亚油酸之类的脂类的方法。从真菌中含有的脂类混合物中纯化得到γ-亚油酸。
DE3603000A1(Milupa)公开了一种高聚不饱和脂肪酸混合物,及其作为婴儿配方食物中脂肪组份的用途。这种脂肪混合物中含有高含量的ARA和二十二碳六烯酸(DHA),两者之比为2.5∶1,还有大量胆固醇。列举的脂肪酸来源是某些类型的巨藻,鱼油,来自牛肉和猪肉的器官脂肪或高度提纯的蛋黄油。DHA和ARA的一种来源据说是褐藻门和红皂门的巨藻。从未有人提出将微生物用作油脂来源。藻类和鱼油通常也含有抑制ARA体内合成的EPA。此外,高度提纯的蛋黄油并不是一种经济的ARA来源。而且,其中没有有关用于补充已有婴儿配方食物的含高浓度ARA添加剂的内容。
所以,一直以来需要一种经济的,工业上可行的,最好不含EPA副产物的ARA生产方法。本发明的目的之一是满足这一需求。
本发明的目的之二是提供用于婴儿配方食物的添加剂和这种添加剂的来源,用以使这些配方食物中的ARA含量接近母乳中的含量。
本发明另一目是提供含ARA的真菌油,可用在肠用,非肠用或皮肤用产品中。
发明概述
本发明涉及含花生四烯酸的真菌油(ARASCO)的生产与使用,以及含有这类油的组合物。可将这类油称为单细胞油。在产油条件下培养真菌,收获真菌并萃取和回收油脂。这些油不需要进一步的化学改性即直接用于为需要ARA的人,包括新生儿,孕妇或哺乳期妇女,或是表现出ARA不足性疾病的人提供补充的ARA。本发明的优点在于生产方便,纯度高,而且没有检知量的EPA。
优选实施方式的详细说明
“ARA”和“EPA”在本文中还分别指花生四烯酸和二十碳五烯酸的残基,当这些残基与甘油经酯化反应成为脂肪酰甘油三酯或磷酸酯的组成部分时。在本文中,当将组合物用作营养补充时,其中EPA的残留含量低于会抑制ARA合成的含量,则该组合物“基本上不含EPA”。本发明能够提供经济的花生四烯酸(ARA)来源。
在一种实施方式中,本发明涉及生产含花生四烯酸而基本上不含二十碳五烯酸(EPA)的真菌油(ARASCO)的方法。本文中,“基本上不含”指在油脂中,EPA的含量低于ARA含量的约1/5。这种油,单细胞油,可以以非改性形式直接使用。本文中,“非改性”指没有共价改变脂肪酸或油本身的化学特性。这样,例如对ARASCO或ARA的临时改性,这种临时改性能够在油脂被吸收时恢复,也在本发明的范围之内。
本发明的非改性真菌油提供的甘油三酯其脂肪酸残基中相对较多的是ARA(至少40%的脂肪酸残基是ARA为宜,50%更好),ARA与EPA残基之比也很高(至少5∶1,至少20∶1更好,w/w)。在本发明之前,还从未有过这种来自天然来源的油脂的报道。虽然如此组成的甘油三酯可以化学合成(例如,通过ARA高含量的游离脂肪酸混合物的酯化反应,或以这些脂肪酸混合物的乙酯进行转酯反应),对这些脂肪酸混合物的操作(例如纯化,酯化等)会引入不需要的副产物。相反,本发明的方法能够通过从天然来源中萃取来提供具有要求组成的甘油三酯。
表1. 几种真菌菌属的脂肪酸组成
大多数其脂肪酸已经鉴定过的真菌种类被发现不生产ARA。Weete,J.D.,真菌脂类的生物化学(Fungal Lipid Biochemistry),Plenum Press,N.Y.(1974)。那些生产ARA的真菌种类中,包括先前鉴定的腐霉属在内的许多除ARA之外还生产大量的二十碳五烯酸(EPA)。表1给出了P.insidiosum和其它真菌的脂肪酸结构。出乎意料的是,P.insidiosum能够生产的ARA不含EPA副产物。至于鱼油,食物补充中的高含量EPA将导致对从饮食来源亚油酸(LOA)形成ARA能力的抑制。所以,虽然在本发明方法中可以使用同时生产ARA和EPA的真菌,但是以使用不产生大量EPA的真菌种类为佳。这些较好的菌种包括Pythium insidiosum和高山被孢霉。以上两种都是能够购买到的,而且保藏在Rockville,Maryland的美国典型培养物保藏中心(American Type Culture Collective),保藏号分别为28251和42430。在本文中,P.insidiosum和高山被孢霉被用作代表性的真菌种类。当然,如本文所述生产含ARA和少量EPA甘油三酯的其它真菌种类也在本发明考虑范围内。
本发明实施方式克服的主要问题之一是饮食来源EPA含量提高在儿童体内引起的ARA生物合成抑制。这一问题可以通过提供ARA含量基本与母乳中相同的婴儿配方食物来解决。在母乳中,ARA∶EPA之比典型地为约20∶1。本发明特别考虑了任何一种可提供足量ARA来克服食物EPA副作用的微生物油。较好的是,使用含ARA的油使得ARA∶EPA之比至少约5∶1。更好的是,至少约10∶1,最好至少约20∶1。可见,与EPA含量相比,最终成品中的ARA含量越高,结果越理想。
在本发明方法中,在适合于生产含ARA油的条件下培养真菌。一般,真菌培养技术是本领域技术人员所熟知的,这些技术可以用于本发明方法。例如,可以在摇瓶中进行真菌接种量的深层培养。摇瓶中装有生长培养基,植入真菌菌丝,在往复式摇床上培养约3至4天。
生长培养基的组成可以不相同,但都含有碳源和氮源。较好的碳源是葡萄糖,其含量在约每升培养基10-100克。对于摇瓶培养,通常使用约15g/l。用量可随要求的最终培养物密度而不同。可以使用的其它碳源包括糖蜜,高果糖玉米糖浆,水解淀粉或其它用于发酵的低成本常规碳源。此外,乳糖可用作P.insidiosum的碳源。这样,乳清,作为一种富含乳糖的低成本碳源可被用作底物。本领域熟练技术人员很容易确定这些碳源的合适用量。通常,在培养过程中需要另外加入碳源。这是因为微生物对碳的消耗量很大,以致于一次性加入全部碳源是不可行的。
氮源的提供通常为酵母提取物,其浓度为约每升生长培养基2至15克提取物。较好的是,提供约4g/l。可以使用其它氮源,包括胨,胰化蛋白胨,玉米浸出液,大豆粉,植物蛋白水解物等。本领域熟练技术人员很容易确定这些氮源的加量。氮源可以成批加入,即在培养前一次性全部加入。
在适宜温度下,典型的是25至30℃培养3至4天后,长出的真菌量足以用作常规搅拌发酵罐(STF)的接种物。这种发酵罐是本领域技术人员已知的,而且是可以购买的。可以按分批、半连续或连续发酵模式进行发酵。较好的是,STF配有船用搅拌桨,但也可使用Rushton式涡流搅拌桨。
添加要求的碳源和氮源来准备发酵罐。例如,可通过每升自来水混合以50克葡萄糖和15克酵母来准备1.5升发酵罐。如前所述,可以使用其它碳源和氮源及其混合物。
含有营养液的反应器需要灭菌,例如通过在接种前的加热。在冷却至约30℃后,可以加入接种物,并开始培养。利用空气喷射来进行气体交换。空气的喷射速度可以不同,但是以调节至约0.5至约4.0VVM(每分钟单位体积发酵罐的空气体积)为宜。较好的是将溶解氧浓度维持在溶液饱和氧浓度的约10%至约50%。所以,在培养过程中可能需要对喷射速度进行调节。需要进行搅拌。由搅拌桨进行搅拌。搅拌的桨外缘速度宜设定在约50cm/sec至约500cm/sec,约100至200cm/sec更好。
通常,接种量可以不相同。典型的是使用约2%至约10%体积的接种物。较好的是,可以在发酵罐中使用约5%体积的种子菌株。
必须对营养物水平进行监测。当葡萄糖水平下降至低于5g/l时,应该另外添加葡萄糖。一个典型的培养周期使用每升约100克葡萄糖和约15克酵母提取物。在培养过程中要求将氮源耗尽,因为这样能够促进真菌生产油脂。这在以高山被孢霉作为生产微生物时尤为如此。
在一种特别好的实施方式中,可以对包含ARA的油脂含量很高的高山被孢霉使用很高的营养物含量进行发酵罐培养。出人意料地发现,可以在发酵开始时使用高于15g/l酵母提取物所提供的量的含氮营养物浓度,只要在发酵过程中加入的含碳营养物总量也相应较高。含碳营养物总量,宜在发酵的前25-50%时间内连续或间歇地加入,或者在上述时段内的不同时刻等量添加,以相当于每升培养基75-300g葡萄糖(C∶N≥5∶1,按w/w葡萄糖∶酵母提取物表示)为宜。在一具体的实施方式中,氮源是大豆粉,加量约每升培养基16克,碳源在开始时的含量相当于约80或80克以上葡萄糖。使用高含量的碳氮营养物时,最好对含有两种营养物的溶液分别灭菌。还发现,对于高含量碳源发酵来说,部分限制氮源,然后在发酵的过程中连续,或一次或分多次加入其余氮源能够提高生物质产量。
有时,培养物会产生过量的泡沫。可以可选性的添加本领域技术人员熟知的消泡剂,例如Mazu 310R或植物油来防止泡沫。
发酵温度可以有所不同。但是,同时生产ARA和EPA的真菌在较高温度培养时倾向于生产较少的EPA和较多的ARA。例如,高山被孢霉在18℃以下培养时开始产生EPA。所以,宜将温度维持在诱导ARA优先性生产的值。合适的温度一般为约25℃至约30℃。
较好的是,将培养持续至要求的生物质密度。要求的生物量是约25g/l的微生物。这样的生物量一般可在接种后48至72小时内达到。此时,生物质通常含有约5至40%复合脂,即油脂,其中甘油三酯的组成中含约10-40%是ARA残基,或者至少40%更好,至少50%还要好,此时可以收获生物质。
本发明的产ARA真菌发酵可以在约pH5至8的发酵培养基中进行。但是,限制培养基的pH而不是任其无限制的上升可以提高高山被孢霉培养物的生物质、油和ARA产量。在发酵过程中维持高氧水平也可以提高产量。在发酵罐中使用高营养物含量时,对发酵过程的这些改进尤为有效。
最初的氮源水平超过约15g/l酵母提取物当量,和/或碳源超过约150g/l葡萄糖时,真菌的生长会受到抑制。可以通过分批发酵来克服这种生长抑制,例如,将发酵所需的营养物分成几份,顺次在先前加入的营养物部分或全部被代谢耗尽时先后加入。可以通过只加入碳源来克服生长抑制(参见Shinmen等)。还发现,可以通过将全部营养物分成等份后在发酵过程中加入,或连续加入营养物溶液来获得上述效果。同样,还出乎意料地发现,只在最初碳源水平很高的发酵反应中加入氮源可以获得上述效果。
出乎意料的是还发现,限定发酵的pH,维持高的氧分压,或两者结合可以缓解生长抑制。在高营养物浓度、低pH(pH=5-6)下进行的高山被孢霉发酵可促进生物体的生长(同时提高油脂产量)。但是,这种条件下产出的油,其中的ARA残基含量较低。相反,高pH(pH=7-7.5)发酵提高油脂中的ARA含量,但生长情况较差。在较好的方式中,本发明的方法涉及pH的限定,即pH在发酵的早期较低而在后期较高。早期包括快速(指数)生长期,该阶段中营养物代谢迅速;后期包括稳定期,此时,细胞分裂被抑制,通常是由于一种或多种营养物质不足,而富含ARA的油脂的生产得以加强。可以通过在发酵过程中分两次或多次调节pH水平来进行限定。
类似地发现,维持培养基中的高溶氧浓度(D.O.)(如,≥40%空气饱和浓度)能够缓解高营养物浓度引起的生长抑制和/或提高油脂中的ARA残基的相对浓度。D.O.可以通过提高罐压(在发酵罐顶部压入更多的空气),加强搅拌(提高搅拌桨的外缘速度),和提高通气量(即提高给定时间内通过发酵罐的空气量,通常表示为提高VVM,每分钟单位发酵罐体积的空气量)和/或增加喷射气体中O2含量。在此条件下的发酵被发现提高了碳利用率,结果在发酵罐中得到较高的生物质浓度和较大的富ARA油脂生产能力。具体地说,进行一项或多项上述改进的发酵可生产至少含40%ARA残基的可萃取甘油三酯类油脂,至少50%ARA残基更好。
在特别好的实施方式中,发酵培养基含有相当于≥80g/L葡萄糖的碳源和相当于≥16g/L酵母提取物的氮源,灭菌后将培养基调至pH5至6之间。接种后,调节培养基的pH等于或略高于最初的水平。当碳源水平下降至≤60g/L葡萄糖当量时(通 常约48小时后),将pH调节的设定值改为pH≥6。在耗氧速度(和/或二氧化碳释放速度CER)达到或接近最大时(通常约72小时后),以约0.1pH/小时的速度将设定值升高至pH6.5至pH7之间。然后在发酵的最后阶段,将pH控制在约pH=7-7.5以下。
对这一实施方式来说,培养基中的溶氧浓度(D.O.)被维持接近或高于空气饱和浓度的40%,最好是通过逐渐升高罐压至11psi,加强搅拌至约300cm/sec搅拌桨外缘速度,和提高通气速度至约0.5体积/发酵罐体积/分钟。在一段快速生长期及O2高消耗发酵后,生长速度(O2消耗)下降。此时可减弱搅拌/通气,只需维持高水平的D.O.,通常约40%空气饱和浓度以上。
通过如上所述优化高山被孢霉发酵,能够获得很高的生物质产量,其中约含20-60%的油脂,油脂中按重量计25-70%是甘油三酯形式的ARA残基。可以如本文所述收获生物质(和油)。较好的是,可以在达到最大生产能力的48小时内,根据gARA/L/天测定,收获生物质。
可以用各种合适的方法进行收获,例如过滤、离心或喷雾干燥。过滤因为其成本低而被优选。
收获后,可以对菌丝饼进行萃取。菌丝饼指收获后得到的生物质集合。菌丝饼可以是松散的或压实的,粉碎的或未粉碎的。可任选的是,在萃取前,菌丝饼可以通过例如真空干燥、流化床干燥、喷雾干燥或冷冻干燥完全脱水。如果选择这样做,以使用非极性溶剂来萃取含ARA的油脂为宜。虽然任何非极性溶剂都适用,但以己烷为佳。
在一较好的实施方式中,用无水己烷湿磨或渗滤来从干生物质中萃取油脂。通常以溶剂∶生物质约为5∶1(w/w)的比例加入溶剂。湿磨后,通过倾析或离心将固体与滤液分离。最好保持含溶剂的萃取液(胶束)无氧,以防止油脂中不饱和脂肪酸残基的氧化。胶束经去溶剂化后生成粗真菌油。
以非极性溶剂萃取自真菌生物质的粗油可能是浑浊的,尤其是在将生物质磨碎时,因为磨碎可能释放出例如细胞壁碎片和可溶性多糖之类的细小颗粒。这种浑浊油脂的澄清可以通过将粗油溶解在极性较强的溶剂中,例如丙酮或醇中。在一较好的实施方式中,萃取自真菌菌丝的粗油进一步经丙酮萃取/沉淀澄清化。可在浑浊的粗油萃取液中加入丙酮(以使油含量约20%为佳,即每体积粗油加入4体积丙酮),充份混合,然后将混合物静置足够长的时间以沉淀出细小颗粒(通常为室温下1小时)来制备丙酮胶束。利用离心和/或过滤来澄清含油的丙酮胶束,然后除去溶剂将生成经丙酮澄清的真菌油。最好将经丙酮澄清的真菌油用于后加工(如利用常规技术脱胶,脱色和除臭),因为萃取真菌生物质过程中的细小颗粒如果不在丙酮处理步骤中被去除,会干扰精炼加工。
另一优选实施方式涉及逆流萃取干生物质,这种萃取可以在市售的萃取单元中进行,例如Crown Ironworks(Crown Mark IV)或French Inc.的产品,这些通常不用于萃取植物油,而是用于萃取灰尘和污泥。虽然,由于没有将生物质重新粉碎使得萃取效率不高,但逆流萃取过程的优点在于产生的细小颗粒较少,因而降低了回收澄清的精制油的技术难度。
或者,湿的菌丝饼(一般含约30至50%的固体)可在粉碎后直接用极性溶剂,例如乙醇或异丙醇萃取,或用CO2或NO之类的溶剂进行超临界流体萃取。最好在萃取前将菌丝饼粉碎。本发明的优点在于能够经济的利用超临界流体萃取技术。McHugh等,超临界流体萃取(Supercritical Fluid Extraction),Butterworth(1986)。这些技术是本领域技术人员熟知的,其中包括现在用于对咖啡豆脱咖啡因的那些技术。
一种较好的水溶液萃取方法涉及在一合适的反应釜中将菌丝体与极性溶剂异丙醇混合。这样的反应釜是已知的。对每份生物质使用3至6份溶剂为宜。最好,在氮气氛下或在抗氧化剂存在下混合,以防止脂萃取液中ARA的氧化。本文中,“脂萃取物”,“油”,“脂复合物”和“真菌油”被互换使用。
萃取后,可以过滤混合物以从含有脂萃取物的溶剂中滤出生物质。此时,可回收生物质用作食物补充剂。本文中,“食物补充剂”指可以提供给动物的,与谷类等一般饲料混合的饲料或添加剂。
将溶剂与脂萃取物分离,并且也可以将其回收再利用,例如通过蒸发到合适的收集器中,而留下本文所称的“粗油”。使用异丙醇作为溶剂较好地去除了粗油中的所有水份,因为蒸发去除了自然形成的水/异丙醇共沸混合物。
虽然粗油可以不经进一步处理而被使用,但是也可以将其进一步纯化。在这一附加纯化步骤中,可以使用本领域技术人员已知的,用于从植物性产物中制备卵磷脂的方法。这些方法对含ARA的质或ARA本身并不进行化学或共价改性。
产量并不一定,但一般每100克干菌丝产约5克含ARA的磷脂。在使用高山被孢霉时,每100克干菌丝还可以再获得10-15克甘油三酯。粗油或精制产品都可给人使用。以上两者都包括在本文的ARASCO定义之内。
本发明的最佳目的是提供一种用于婴儿配方食物的添加剂,使得这种配方食物中的ARA含量接近母乳中的ARA含量。表2将ARASCO中的脂肪酸组成与母乳中的,以及含有和不含ARASCO的婴儿配方食物中的进行了比较。
表2 真菌油产品与母乳中脂肪酸组成的比较
脂肪酸 ARASCO 婴儿配方食物1 配方食物+油 母乳
8:0 -- 24.1 23.6 0.35
10:0 -- 17.7 17.3 1.39
12:0 -- 14.9 14.6 6.99
14:0 4.6 5.8 5.8 7.96
16:0 16.0 6.8 7.0 19.80
16:1 3.2 0.2 0.3 3.20
18:0 -- 2.3 2.3 5.91
18:1 26.4 10.0 10.3 34.82
18:2n6 9.9 17.4 17.3 16.00
18:3n3 4.1 0.9 1.0 0.62
20:1 2.2 0.1 0.14 1.10
20:2n6 -- -- -- 0.6 1
20:3n6 1.4 -- 0.03 0.42
20:4n6 32.0 -- 0.64 0.59
20:5n3 -- -- -- 0.03
22:1 -- -- -- 0.10
22:4n6 -- -- -- 0.21
22:5n6 -- -- -- 0.22
22:6n3 -- -- -- 0.19
1 Simopoulis,A.,w-3酸与健康与疾病(Omega-3 Acids in Health and Disease),pp.155-156(1990)。
可以看到,用ARASCO补充的婴儿配方食物中的ARA含量与母乳中的ARA水平很接近。此外,加入ARASCO并没有明显改变婴儿配方食物中总脂肪酸组成。通常,每升婴儿配方食物可使用约50至1000mg的ARASCO。具体的ARASCO需要量取决于其中的ARA含量。该含量可以占油中脂肪酸的约10%至约70%。但通常ARA含量为约30至50%。较好的是,用以补充婴儿配方食物的油含有至少40%ARA脂肪酸残基,至少50%更好。ARA含量约30%时,较好的补充配比是每升婴儿配方食物约600至700mg的ARASCO。这样的配比仅以一份ARASCO对50份配方油脂的比例稀释了婴儿配方食物中原有的脂肪成份,例如 (RossLaboratories,Columbus,Ohio)。对于ARA含量更高的油也可计算得到类似的稀释比。ARASCO以基本不含EPA为宜。
在上述方法中使用Pythium insidiosum时,萃取得到的含ARA的油脂主要是磷 脂。但是已经发现,从如本文所述进行培养的Pythium insidiosum也可以回收到大量高ARA残基含量的甘油三酯。在所述方法中使用高山被孢霉时,含ARA的油主要是甘油三酯。两种形式的ARASCO都被用作婴儿配方食物的添加剂。前者不仅为配方食物提供ARA,而且还提供乳化剂,即通常加在市售配方食物中的卵磷脂。高山被孢霉产生的油脂似乎生产起来更经济。
除了用作婴儿配方食物添加剂之外,本发明的含ARA油脂还有许多其它用途。正如本领域技术人员已知的,许多疾病与缺乏ARA有关,例如消瘦(Vajreswari等,代谢(Metabolism)39:779-782(1990)),皮肤病(Melnik,Bo,Monatsschr.Kinderheilta,138:162-166(1990)),肝病,苯丙酮酸尿症,精神分裂症,慢性记忆障碍或各种过氧化酶疾病。在本发明的一种实施方式中,通过使用药物有效量的本发明的油脂治疗上述疾病。药物有效量一般指使得病人体内的ARA血清水平达到正常所需的量。用于补充治疗这些疾病尤其好的是上述高ARA含量的油,尤其是含至少40%ARA的油,50%ARA残基更好。根据保健医师来选择,油脂可以经肠给药,外用或非经肠给药。
本领域技术人员已知的胶囊化是一种经肠给药的有效方法。可以给需要饮食补充ARA的人服用含有真菌油的胶囊。该方法对给孕妇或哺乳期妇女服用ARA尤为有效。
在使用ARASCO治疗与缺乏ARA有关的疾病时,应该使用药物有效量。本领域技术人员不经毒性实验就可以确定这一用量。通常,该量是0.5-2.0g/天,一般能够使ARA血清水平达到正常。
本发明的其它实施方式提供了含有ARASCO,例如本文所述的高ARA含量的油的化妆品组合物。化妆品组合物指用作化妆品的那些化合物。这类组合物一个较好的实例是防皱霜。这类化妆品组合物提供了对皮肤局部使用ARA以有助于保持皮肤弹性的有效方法。
以上对本发明进行了全面的描述,以下的非限定性实施例用于进一步说明本发明。
实施例1.制备P.insidiosum脂并加入婴儿配方食物
在一80L(约略体积)的发酵罐中,混合51L自来水,1.2kg葡萄糖,240g酵母提取物和15ml MAZU 消泡剂。发酵罐在121℃灭菌45分钟。在灭菌过程中另加入5L冷凝水。将pH调至6.2,然后加入约1L(细胞密度为5-10g/l)的Pythiuminsidiosum(ATCC#28251)接种物。将搅拌速度调节至125RPM(250cm/sec外缘速度),通气速度设定为1SCFM(标准立方英尺/分钟)。第24小时,将通气速度增加至3SCFM。第48小时,另外加入50%葡萄糖浆2L(1kg葡萄糖)。第50小时,放 罐收获,产量约2.2kg湿重(约15g干重)/L。将收获后的生物质在吸滤机上挤压成高固含量(50%固体)的饼状,然后冷冻干燥。用研钵和研杵粉碎干燥后的生物质,每200g干生物质用1L己烷在室温下连续搅拌萃取2小时。然后过滤混合物,蒸发滤液,每100g干生物质得约5-6g粗油。然后再将每20g干生物质用1L的乙醇在室温下萃取1小时,过滤后蒸发掉溶剂,每100g干生物质另外得到22g粗油。第二份中主要是磷脂,而第一份含磷脂和甘油三酯的混合物。合并后得到的油含约30-35%花生四烯酸,没有检知量的EPA。可将这种油滴加到市售的婴儿配方食物产品 (Ross Laboratories,Columbus,Ohio)中,补充比为每L已制备基质含60mg。
实施例2.制备高山被孢霉脂并加入婴儿配方食物
在一个2L摇瓶中培养高山被孢霉(ATCC#42430),前者含有1L自来水和2g土豆葡萄糖培养基。摇瓶保温于25℃回转搅拌7天。离心收获生物质后,将其冷冻干燥,得约8g富含脂体的菌丝。如实施例1所述用己烷萃取菌丝,得到约2.4g粗油。此油中含有约23%花生四烯酸,按100mg/L的浓度加入到市售的婴儿配方食物产品 中。
实施例3.利用高山被孢霉大量生产花生四烯酸
在含有GYE培养基(50g/L葡萄糖和6g/L Tastone 154)的接种罐中接入高山被孢霉。发酵温度设定在28℃,最初搅拌速度为130-160cm/sec,最初罐压为6psi,最初通气速度为0.25VVM。在灭菌前将pH调节至5.0,灭菌后的最初发酵pH调节至5.5。用8N的NaOH维持培养基的pH≥5.5。按以下顺序调节搅拌/通气,将氧水平维持在D.O.≥40%:将罐压增加至11psi;提高搅拌的桨叶外缘速度至175cm/sec;将通气速度提高至0.5VVM。根据需要加入Dow 1520-US来控制泡沫。(在灭菌前需加入约0.1ml/L消泡剂来帮助防止发泡)。
pH升至6.0以上后的12小时内,将接种物从种子发酵罐转移到主发酵罐中。
主发酵罐含有GYE培养基(50g/L葡萄糖和6g/L Tastone154);葡萄糖单独灭菌,灭菌后加入主发酵罐中。将发酵温度设定在28℃,最初搅拌速度为160cm/sec,最初罐压为6psi,最初通气速度为0.15VVM。灭菌后的最初pH设定在5.5,用8N的NaOH维持培养基的pH≥5.5。在稳定期(接种后约24小时以后开始)允许pH升高,但通过添加H2SO4维持在pH6.8以下。通过逐步地将罐压增加至11pis,提高搅拌的桨叶外缘速度至175cm/sec,将通气速度提高至0.5VVM来将氧水平维持在D.O.≥40%。根据需要加入Dow 1520-US来控制泡沫。(在灭菌前需加入约0.1ml/L消泡剂来帮助防止发泡)。
培养物每12小时采样作生物质及脂肪酸分析。在pH升至6.5以后的3-4天开始收获。干燥的生物质密度应≥8.5g/L。在发酵液中葡萄糖浓度应从50g/L下降至25g/L。在收取时,全部培养的发醇液用盒式离心机从废培养基中分离出菌丝体,并将此生物质干燥。
实施例4.高山被孢霉产生的生物质的产量提高-第一批
根据实施例3的方法,接种以摇瓶中的培养物,将高山被孢霉培养在20L的搅拌发酵罐中。在65g/L葡萄糖(Staleydex),和6g/L酵母提取物(Tastone 154)培养高山被孢霉产生12g/L生物质。在第16小时另外加入6g/L Tastone 154,结果产生18g/L生物质。
实施例5.高山被孢霉产生的生物质的产量提高-第二批
为了通过追加Tastone 154来进一步增加生物质而进行了一系列实验。这些实验为2×20L发酵,停留时间为168小时。两者的最初葡萄糖浓度都是100g/L(与实施例4的65g/L相比)。一罐添加3×6g/L的Tastone 154,另一罐添加4×6g/L。将酵母提取物配制成浓溶液,高压灭菌,在灭菌后的不同时刻加入。
为了制备接种物,将使用的种子(1ml浸渍后的菌丝)接种在2个含50ml GYE培养基(100g/L Staleydex,6g/L Tastone 154)的烧瓶中,在28℃、150rpm培养4天。生长4天后,发酵液中含有球结的生物质;球的直径为2-5mm。在这些烧瓶中的生长比预计的慢,可能是由于高葡萄糖浓度。生物质在Waring混合仪中浸渍2×3秒,取25ml浸渍液接种到2个2.8L的Fernbach接种烧瓶中,净体积为800ml。(在先前的实验中曾使用10ml的浸渍液。提高接种量是因为种子瓶中生物质的密度较低,而且估计在Fernbach中的生长会因高葡萄糖浓度而较慢。)Fernbach中的培养基为葡萄糖(Staleydex)100g/L和酵母提取物(Tastone 154)8g/L。葡萄糖和酵母提取物分别经40分钟灭菌。种子的发酵温度维持在28℃,搅拌速度维持在100至150rpm。
在Fernbach烧瓶中培养44小时后,接种物被转移至2个20L的发酵罐中。接种物的形式为十分松散的菌丝聚集体,生物质密度约5.2g/L。
含有1.6kg(10%)葡萄糖(Staleydex)和Mazu204消泡剂(1.6g,溶解在12.5LR.O.H2O中)的14和15号发酵罐在122℃灭菌45分钟。然后在每个罐中(在0时刻)加入800ml(5%)接种物。发酵罐的操作参数如下:
温度:28℃,
pH:用2N NaOH和2N H2SO4维持在5.5,
通气:0.5VVM,
背压:0.2bar
搅拌(最初):80cm/sec,
D.O.:控制在40%以上。
14号:3×6g/L Tastone 154
将酵母提取物(Tastone 154)溶解成96g/L的浓度并高压灭菌1小时。分3次在第0,20和26小时每次加入1L(1.8%)。
第15小时,DO跌至40%以下,从第15小时至第22小时将搅拌速度升高至175cm/sec。然后通过用氧气改变气流量来调节DO;从第23小时至72小时,在气流中加入氧气。从第36小时开始,进一步提高搅拌速度以保证充分混合。至第48小时,搅拌速度已升高至200cm/sec;至第72小时,升高至250cm/sec;至第80小时,升高至280cm/sec。在第120小时,将搅拌速度提高至290cm/sec以加强温度控制。在第144小时,将搅拌速度降低至280cm/sec。
15号:4×6g/L Tastone 154
将酵母提取物(Tastone 154)384g溶解成96g/L的浓度并高压灭菌1小时。分4次在第0,20,26和32小时每次加入1L(2.4%)。
第16小时,DO跌至40%以下,将搅拌速度升高至175cm/sec,直至第23小时。然后通过用氧气改变气流量来调节DO于40%以上;从第23小时至72小时,在气流中加入氧气。从第36小时开始,进一步提高搅拌速度以保证充分混合。至第48小时,搅拌速度已升高至210cm/sec;至第72小时,升高至260cm/sec;至第80小时,升高至290cm/sec。在第90小时,将搅拌速度降低至280cm/sec。在第144小时,将搅拌速度降低至260cm/sec。
过程观察:
在接种时,两罐中的生物质都是十分松散的羽毛状菌丝聚集体。至24小时,开始形成球。球很小(1-3mm),具有小的内核和宽而松的外缘。在第48小时,球体变大,形状更清晰。至72小时,外缘变窄,出现许多松散的菌丝片段,这说明球开始解体。至168小时,球核直径达0.5至2mm,外缘因菌丝聚集而缩成粗线,并且形成了许多菌丝聚集体。
发酵罐只在最初的24小时略微发泡。此后,泡沫量增加,并通过在泡头高于2-4cm时人工加入消泡剂得以控制。至48小时前,泡沫略有下降,但时有间歇性的爆发。在发酵过程中,两罐的泡沫都有一次到达出口滤网。发酵约需150ml消泡剂。
两个发酵罐的头部都积累了大量增殖的生物质。对于利用表面积/体积比很大的小罐进行真菌发酵来说,这是一个常见问题。在罐15中,增殖生物质量似乎是在最后的24小时内增加的,期间,体积下降导致了大量的爆溅物(液位接近搅拌桨的 顶部)。168小时后发酵罐中的最终体积是13L。
显微镜检显示,至72小时,在培养物发酵液中有许多碎片,而且发现有损伤和萎缩的真菌端部。在第168小时的尼罗红染色显示在细胞质中有油滴。与有时可见的大油滴相比。油滴很小但很多,表3显示了生物质和油产量,以及碳氮源的利用率。
表3 发酵过程
实施例6.高山被孢霉产生的生物质的产量提高-第三批
这一组实验是为了通过增加磷酸盐和无机盐的水平来进一步提高产量。过程与实施例5基本相同,但葡萄糖和消泡剂Mazu204溶解在11.5L而不是12.5L的R.O.H2O中,为在第30小时添加的盐溶液留出空余体积。14号罐添加Fe,Zn,和Cu;罐15添加Fe,Zn,和Cu的磷酸盐。
罐14:3×6g/L Tastone 154
将酵母提取物溶解成96g/L浓度,分成1L的3份,高压灭菌1小时。在第0,22和28小时加入1L等份。在第22和28小时,二氧化碳释放速度(CER,指示发酵罐中的代谢速度)呈指数增长,发酵液开始需要加碱。
加入的盐包括:
FeCl36H2O 480mg
ZnSO47H2O 240mg
CuSO45H2O 16mg
将FeCl3溶解在1L 5g/L的柠檬酸溶液中。加入其余的盐,用NaOH将pH调节至4.5。将溶液高压灭菌1小时。在第30小时将盐加入。
发酵罐最初的搅拌速度是50cm/sec而不是原先计划的80cm/sec,因为发酵罐中的最初液位(13L)刚好浸没搅拌桨的端部,较高的搅拌速度会导致严重的喷溅。在第1 6小时,D.O.降至40%以下,由此至第28小时将搅拌速度提高至175cm/sec。然后用氧气改变气体流量来将D.O.控制在40%以上。在第46小时,为了混合而将搅拌速度提高至190cm/sec。搅拌速度被进一步升高,第48小时为200cm/sec,至51小时为220cm/sec,至53小时为235cm/sec,至56小时为250cm/sec,至57小时为260cm/sec,至70小时为280cm/sec。即使在这样的搅拌速度(450rpm)下,混合情况仍然较差。虽然维持着最低标准的“略有移动”,但生物质的翻动非常缓慢,有些地方几乎静止。加入数滴消泡剂降低了泡头,消除了静止区。在第116小时,将搅拌速度降低到265cm/sec,在第1 20小时,进一步降低到250cm/sec。
发酵罐在约第18小时开始产生泡沫。泡沫通过人工加入消泡剂来控制。第一次加入消泡剂在第20小时。至24小时,发酵反应大量产生泡沫,需要经常性的添加消泡剂。至72小时,大多数泡沫下降。但是,发酵反应仍然需要在有时加入消泡剂。
至24小时,生物质呈非常松散的球状(1-2mm)和松散的菌丝聚集体。有大量的细胞碎片存在。至48小时,生物质呈十分松散的菌丝聚集体,很小的球状(1-2mm),球的内核很小而外周松散,以及没有松散外周的紧缩球状(1-3mm)。至96小时,生物质成紧缩园球状(1-2mm),针状颗粒(0.5mm以下)和疏松的菌丝聚集体。第144小时的尼罗红染色显示在菌丝中存在着大量非常小的油滴。
罐15:3×6g/L Tastone 154
将酵母提取物溶解成96g/L浓度,高压灭菌1小时。在第0,22和26小时3次加入1L等份。在第22和26小时,二氧化碳释放速度呈指数增长,发酵液开始需要加碱。
加入的盐包括:
KH2PO4 77g
FeCl36H2O 480mg
ZnSO47H2O 240mg
CuSO45H2O 16mg
将FeCl3溶解在500ml 5g/L的柠檬酸溶液中。加入其余的盐,用NaOH将pH调节至4.5。将KH2PO4溶解在500ml R.O.水中。将两种溶液高压灭菌1小时,然后冷却至23℃,然后在第30小时混合加入发酵罐。
发酵罐最初的搅拌速度是50cm/sec而不是原先计划的80cm/sec,因为发酵罐中的最初液位(13L)刚好没过搅拌桨的端部,较高的搅拌速度会导致严重的喷溅。在第16小时,D.O.降至40%以下,由此至第27小时将搅拌速度提高至175cm/sec。然后用氧气改变气体流量来将D.O.控制在40%以上。在第41小时,为了进行至少最小量的混合而将搅拌速度提高至200cm/sec。搅拌速度被进一步升高,第42小时为220cm/sec,至46小时为230cm/sec,至51小时为235cm/sec,至70小时为240cm/sec。即使在这样的搅拌速度(410rpm)下,混合情况仍然为较差至中等。维持着最低程度的生物质移动。在第80小时,将搅拌速度降低到205cm/secc。
发酵罐在约第18小时开始产生泡沫。泡沫通过人工加入消泡剂来控制。第一次加入消泡剂在第17小时。至20小时,发酵反应大量产生泡沫,需要经常性的添加消泡剂。至72小时,大多数泡沫下降。但是,发酵反应仍然需要在有时加入消泡剂。
至24小时,生物质呈非常松散的球状(1-2mm)和松散的菌丝聚集体。有大量的细胞碎片存在。至48小时,生物质呈十分松散的菌丝聚集体,很小的球状(1-2mm),球的内核很小而外周松散,以及没有松散外周的紧缩球状(1-3mm)。至96小时,生物质呈园球状,直径1-2mm,其中许多具有松散的发丝状的外周,以及许多疏松的菌丝片段。第144小时的尼罗红染色显示在菌丝中存在着大量非常小的油滴,而且在其它菌丝充满了很大的油滴。
罐15与罐14的区别仅在于添加了磷酸盐的罐15在整个发酵过程中,在较低的搅拌速度下显示出较好的混合情况。罐15中生物质的形态更“松散”。表4列出了生物质和油脂的产量,以及碳的利用率。高磷酸盐含量发酵的特征在于,高葡萄糖利用率(罐15为82g/L,而罐14为64g/L),高生物质积累和存在于部分菌丝 中的大油滴。
表4 发酵过程
实施例7.大规模生产含有花生四烯酸的高山被孢霉生物质
从增殖罐向含有GYE培养基(50g/L葡萄糖和6g/L Tastone 154)的种子罐接种。温度维持在28℃,最初搅拌速度设定在130至160cm/sec之间(约43rpm)。最初罐压为6psi,最初通气速度为0.25VVM。灭菌前的pH调节至5.0,灭菌后的最初pH设定在5.5。按照以下顺序将培养基中的氧浓度维持在D.O.≥40%:(i)将罐压升至11psi,(ii)将搅拌速度提高至156-175cm/sec搅拌桨外缘速度,(iii)将通气速度提高至0.5VVM。根据需要添加消泡剂Dow 1520-US来控制泡沫。(为了有助于防止发 泡,应该在灭菌前加入约0.1ml/L消泡剂)。接种后,用8N的NaOH维持培养pH≥5.5。
pH升至6.0以上后的12小时内,将种子罐中发酵液转移至主罐中。主罐培养基含有:
80g/L葡萄糖(ADM)
16g/L大豆粉(ADM nutrisoy)
30mg/L FeCl36H2O(Sigma/Aldrich)
1.5mg/L ZnSO47H2O(Sigma/Aldrich)
0.1mg/L CuSO45H2O(Sigma/Aldrich)
1mg/L生物素(Sigma/Aldrich)
2mg/L盐酸硫胺(Sigma/Aldrich)
2mg/L泛酸(半钙盐)(Sigma/Aldrich)(灭菌前调节至pH4.8-5.0)
用种子罐基质接种主罐(11.8%)。将发酵罐温度维持在28℃。最初搅拌速度设定在162cm/sec(约23rpm),最初罐压为6psi,最初的通气速度为0.15VVM(约300scfh)。
将培养基中的氧浓度维持在D.O.≥40%:(i)将罐压升至11psi,(ii)将搅拌速度提高至300cm/sec搅拌桨外缘速度(约增加了30cm/sec),(iii)将通气速度提高至0.5VVM。
根据以下pH调节方法来控制pH:
将灭菌后的最初pH设定为5.5。用8N的NaOH维持pH≥5.5。
在接种后24至36小时,加入:2g/L KH2PO4(110kg溶于约700L水中)。
第48小时,如果葡萄糖浓度≤60g/L,将pH设定值改为≥6.1。
第72小时,以每小时约0.1pH的速度缓慢地将pH升至≥6.6。
在必要时添加H2SO4维持pH低于7.3。
每隔12小时对发酵罐取样一次进行生物质和脂肪酸的分析,在pH升至≥6.6约3天后(约接种6天后)开始收获。干生物质的密度应≥24g/L。发酵液中葡萄糖浓度应从80g/L下降至≤14g/L。
将全部发酵液通过旋转真空过滤器过滤,将菌丝与废培养基分离,如此进行收获。
根据本实施例进行的两批典型发酵的结果列于表5和表6。
表5 高山被孢霉的发酵过程
表6 高山被孢霉的发酵过程
Claims (22)
1.一种婴儿配方食物,包含真菌产甘油三酯油脂,所述真菌产甘油三酯油脂含有至少40%甘油三酯形式的花生四烯酸(ARA),且二十碳五烯酸(EPA)含量不超过ARA的十分之一,所述真菌产油脂是高山被孢霉产生的。
2.如权利要求1所述的婴儿配方食物,所述真菌产甘油三酯油脂包含至少50%甘油三酯形式的ARA。
3.如权利要求1或2所述的婴儿配方食物,所述真菌产油脂含有不超过ARA二十分之一的EPA。
4.如权利要求3所述的婴儿配方食物,所述真菌产油脂基本上不含EPA。
5.如权利要求1或2所述的婴儿配方食物,其中含ARA甘油三酯的含量与母乳中的相当。
6.如权利要求1或2所述的婴儿配方食物,其中的油脂是非改性油脂。
7.如权利要求5所述的婴儿配方食物,其中的油脂是非改性油脂。
8.一种生产含至少40%甘油三酯形式的花生四烯酸(ARA)且二十碳五烯酸(EPA)含量不超过ARA的五分之一的真菌产甘油三酯油脂的方法,包括在发酵罐中在高营养培养基中有氧半连续培养高山被孢霉使之生产含ARA的真菌产油脂,所述高营养培养基含相当于每升培养基至少80g葡萄糖的碳源和相当于至少16g/l酵母提取物的氮源,其中按w/w葡萄糖:酵母提取物表示的C:N大于等于5:1,并且,所述培养维持溶氧浓度高于或等于40%空气饱和浓度。
9.一种生产含至少40%甘油三酯形式的花生四烯酸(ARA)且二十碳五烯酸(EPA)含量不超过ARA的五分之一的真菌产甘油三酯油脂的方法,包括在发酵罐中在高营养培养基中有氧半连续培养高山被孢霉使之生产含ARA的真菌产油脂,所述高营养培养基含相当于每升培养基至少80g葡萄糖的碳源和相当于至少16g/l酵母提取物的氮源,其中按w/w葡萄糖:酵母提取物表示的C:N大于等于5:1,并且,所述培养将溶氧浓度维持在足够高的水平以使所得被孢霉生物质含有直径1-2mm的球。
10.如权利要求8或9所述的方法,所述真菌产甘油三酯油脂包含至少50%ARA。
11.如权利要求8或9所述的方法,在发酵期间维持所述溶氧水平。
12.如权利要求8或9所述的方法,至少在生长期维持所述溶氧水平。
13.如权利要求8或9所述的方法,所述真菌产油脂含有不超过ARA十分之一的EPA。
14.如权利要求8或9所述的方法,所述真菌产油脂含有不超过ARA二十分之一的EPA。
15.如权利要求8或9所述的方法,所述真菌产油脂基本上不含EPA。
16.如权利要求8或9所述的方法,其中,所述含ARA的真菌产油脂是以至少1.0g/L/天的平均速度生产。
17.如权利要求8或9所述的方法,其中,所述含ARA的真菌产油脂是以至少2.0g/L/天的平均速度生产。
18.如权利要求9所述的方法,所述被孢霉生物质在培养后收获,并在回收油脂之前进行流化床干燥或喷雾干燥。
19.如权利要求8或9所述的方法,其中,在培养过程中间歇加入的培养基包含添加氮源。
20.如权利要求8或9所述的方法,所述氮源含大豆粉。
21.如权利要求8或9所述的方法,将被孢霉培养至生物质密度至少为25g/l。
22.如权利要求8所述的方法,至少在耗氧速度最大时段维持所述溶氧浓度。
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2010
- 2010-03-11 JP JP2010053924A patent/JP5281026B2/ja not_active Expired - Lifetime
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2013
- 2013-02-26 JP JP2013035462A patent/JP2013116123A/ja not_active Withdrawn
Non-Patent Citations (2)
Title |
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"Production of arachidonic acid by Mortierella alpina ATCC32222";Pramod K.Bajpai ET AL.;《Journal of Industrial Microbiology》;19911231;第8卷;第179-186页 * |
JP昭64-38007A 1989.02.08 * |
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