CN111601872A - 包含至少一种具有至少20个碳原子的多不饱和脂肪酸(lc-pufa)的油 - Google Patents
包含至少一种具有至少20个碳原子的多不饱和脂肪酸(lc-pufa)的油 Download PDFInfo
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- CN111601872A CN111601872A CN201880082268.0A CN201880082268A CN111601872A CN 111601872 A CN111601872 A CN 111601872A CN 201880082268 A CN201880082268 A CN 201880082268A CN 111601872 A CN111601872 A CN 111601872A
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Abstract
本发明涉及一种油,其包含至少一种具有至少20个碳原子的多不饱和脂肪酸(LC‑PUFA)。发现含LC‑PUFA的油在储存期间易于通过形成微观晶体而胶凝,最终导致不良的质量和处理性质。在包含至少约25重量%的LC‑PUFA和0.2至5重量%的水分含量的微生物油的情况下,已经特别观察到该问题。出人意料地,发现如上所述的含LC‑PUFA的油在油的残留水分中包含小于约8重量%,优选小于约5重量%的游离脂肪酸,则在常规的储存条件下有效地稳定且不显示胶凝性质。因此,本发明涉及包含至少约25重量%的LC‑PUFA和如下的水分含量的油,所述水分含量包含小于约8重量%,优选小于约5重量%的游离脂肪酸。
Description
本发明涉及一种包含至少一种具有至少20个碳原子的多不饱和脂肪酸(LC-PUFA)的油。
基于碳链的长度和饱和特征将脂肪酸分类。基于链中存在的碳数,脂肪酸被称为短链、中链或长链脂肪酸,当在碳原子之间不存在双键时,被称为饱和脂肪酸,而当存在双键时,被称为不饱和脂肪酸。当仅存在一个双键时,不饱和长链脂肪酸为单不饱和的,而当存在多于一个双键时,为多不饱和的。
多不饱和脂肪酸(PUFA)基于从脂肪酸的甲基端的第一个双键的位置分类:ω-3(n-3)脂肪酸在第三个碳原子上包含第一个双键,而ω-6(n-6)脂肪酸在第六个碳上包含第一个双键。例如,二十二碳六烯酸(“DHA”)是链长为22个碳和6个双键的ω-3长链多不饱和脂肪酸(LC-PUFA),通常称为“22:6n-3”。其他ω-3LC-PUFA包括称为“20:5n-3”的二十碳五烯酸(“EPA”)和称为“22:5n-3”的ω-3二十二碳五烯酸(“DPA n-3”)。DHA和EPA已被称为“必需的”脂肪酸。ω-6LC-PUFA包括称为“20:4n-6”的花生四烯酸(“ARA”)和称为“22:5n-6”的ω-6二十二碳五烯酸(“DPA n-6”)。
ω-3脂肪酸是生物学上重要的分子,由于它们存在于细胞膜中,可影响细胞生理,调节生物活性化合物的产生和基因表达,并充当生物合成底物。
亚麻籽油和鱼油被认为是ω-3脂肪酸的良好饮食来源。亚麻籽油不含EPA、DHA、DPA或ARA,而含有亚麻酸(CI 8:3n-3),其是使人体能够制造EPA的组成部分。然而,存在新陈代谢的转化速率可能会很慢和可变,尤其是在健康受损的那些人中的证据。鱼油在脂肪酸组成的类型和水平方面有很大差异,这取决于具体物种及它们的饮食。例如,通过水产养殖饲养的鱼会具有比野生鱼中的ω-3脂肪酸更低含量的ω-3脂肪酸。此外,鱼油具有包含环境污染物的风险且可能与稳定性问题以及鱼腥味或味道有关。
LC-PUFA可以由微生物在发酵过程中产生。LC-PUFA也可以在植物中产生。例如,WO2006/085672描述了一种用于产生包含微生物生物质的LC-PUFA的方法,可以从生物质中分离根据本发明的微生物油。
本发明的微生物油是包含至少约35重量%的三酰基甘油级分的“原油”或“精制油”。“原油”是在不进一步加工的情况下从微生物的生物质提取的油。“精制油”是通过用精制、漂白和/或除臭的标准工艺处理原油获得的油。例如参见美国专利号5,130,242,通过引用以其整体并入本文。微生物油还包括如本文所述的“最终油”,其是已经用植物油稀释的精制油。在一些实施方式中,最终油是已经用高油酸向日葵油稀释的精制油。如本文所用的术语“微生物”包括,但不限于术语“微藻”、“破囊壶菌(thraustochytrid)”,和与本文所述的任何微生物相关的分类学分类。
破囊壶菌是破囊壶菌目的微生物。破囊壶菌包括裂殖壶菌属和破囊壶菌属的成员且被认为是ω-3脂肪酸的替代来源,包括DHA和EPA。参见美国专利号5,130,242。由这些海洋异养微生物产生的油通常比相应的鱼油或微藻油具有更简单的多不饱和脂肪酸谱图。Lewis,T.E.,Mar.Biotechnol.1:580-587(1999)。据报道破囊壶菌物种的菌株产生ω-3脂肪酸,占由该生物体产生的总脂肪酸的高百分比。美国专利号5,130,242;Huang,J.等人,J.Am.Oil.Chem.Soc.78:605-610(2001);Huang,J.等人,Mar.Biotechnol.5:450-457(2003)。然而,分离的破囊壶菌可能在产生的LC-PUFA的特性和量上有所不同。
在一些实施方式中,如本文所述的脂肪酸可以是脂肪酸酯。在一些实施方式中,脂肪酸酯包括ω-3脂肪酸、ω-6脂肪酸及它们的组合的酯。在一些实施方式中,脂肪酸酯是DHA酯、EPA酯或其组合。在一些实施方式中,如本文所述的油或其级分被酯化以产生包含脂肪酸酯的油或其级分。术语“酯”是指脂肪酸分子的羧酸基团中的氢被另一个取代基取代。典型的酯是本领域技术人员已知的,对其的讨论由Higuchi,T.和V.Stella在Pro-drugs asNovel Delivery Systems,第14卷,A.C.S.Symposium Series,Bioreversible Carriersin Drug Design,Ed.Edward B.Roche,American Pharmaceutical Association,PergamonPress,1987,和Protective Groups in Organic Chemistry,McOmie ed.,Plenum Press,New York,1973中提供。酯的实例包括甲基、乙基、丙基、丁基、戊基、叔丁基、苄基、硝基苄基、甲氧基苄基、二苯甲基和三氯乙基。在一些实施方式中,该酯是羧酸保护性酯基、具有芳烷基(例如苄基、苯乙基)的酯、具有低级烯基(例如烯丙基、2-丁烯基)的酯、具有低级烷氧基-低级烷基(例如甲氧基甲基、2-甲氧基乙基、2-乙氧基乙基)的酯、具有低级烷酰氧基-低级烷基(例如乙酰氧基甲基、新戊酰氧基甲基、1-新戊酰氧基乙基)的酯、具有低级烷氧基羰基-低级烷基(例如甲氧基羰基甲基、异丙氧基羰基甲基)的酯、具有羧基-低级烷基(例如羧甲基)的酯、具有低级烷氧基羰氧基-低级烷基(例如1-(乙氧基羰氧基)乙基、l-(环己基氧基羰氧基)乙基)的酯、具有氨基甲酰氧基-低级烷基(例如氨基甲酰氧基甲基)的酯等等。在一些实施方式中,添加的取代基是直链或环状烃基,例如C1-C6烷基、C1-C6环烷基、C1-C6烯基或C1-C6芳基酯。在一些实施方式中,该酯是烷基酯,例如甲基酯、乙基酯或丙基酯。在一些实施方式中,当脂肪酸处于纯化或半纯化状态时,将酯取代基添加到游离脂肪酸分子。或者,在三酰基甘油转化为酯时形成脂肪酸酯。
发现含LC-PUFA的油在储存期间易于通过形成微观晶体而胶凝,最终导致不良的质量和处理性质。该问题用包含至少约25重量%的LC-PUFA和0.2至5重量%,特别是0.5至2重量%的水分含量的微生物油特别地观察到。
尽管共享油可能至少在短期内有助于破坏晶体,这会产生不胶凝的油,但仍需要避免在油中形成微观晶体。
出人意料地,发现如果油组合物在油的残留水分中含有小于约8%,优选小于约5重量%的游离脂肪酸,则含LC-PUFA的油在常规储存条件下是有效地稳定且不显示如上所述的胶凝性质。
因此,本发明涉及包含至少约25重量%的LC-PUFA和如下的水分含量的油,该水分含量包含小于约8重量%,优选小于约5重量%的游离脂肪酸。在一些实施方式中,微生物油还包含至少约25重量%的二十二碳六烯酸,至少约10重量%的二十碳五烯酸和小于2重量%、优选小于1重量%的水分含量。
根据本发明的油是衍生自微生物生物体或植物的油。
在现有技术中广泛地描述了包含含PUFA的脂质的微生物生物体。在这种情况下,所用的细胞特别地可以是已经天然产生PUFA(多不饱和脂肪酸)的细胞;然而,它们也可能是由于合适的基因工程方法或由于随机诱变显示PUFA的产生改善或已经完全能够产生PUFA的细胞。PUFA的产生可以是营养缺陷的、混合营养的或异养的。
根据本发明的细胞优选地选自藻类、真菌(特别是酵母)、细菌或原生生物。所述细胞更优选是微生物藻类或真菌。
产油酵母的合适细胞特别是耶氏酵母属(Yarrowia)、念珠菌属(Candida)、红酵母属(Rhodotorula)、红冬孢酵母属(Rhodosporidium)、隐球酵母属(Cryptococcus)、丝孢酵母属(Trichosporon)和油脂酵母属(Lipomyces)的菌株。
产油的微藻类和藻类样微生物的合适细胞特别是选自不等鞭毛门(phylumStramenopiles)的微生物(也称为不等鞭毛门(Heterokonta))。不等鞭毛门的微生物可以特别地选自以下微生物组:汉姆门(Hamatores)、普罗特蒙门(Proteromonads)、欧帕门(Opalines)、德维尔派门(Developayella)、戴普罗弗门(Diplophrys)、拉普利门(Labrinthulids)、破囊壶菌门(Thraustochytrids)、拜尔瑟门(Biosecids)、水霉门(Oomycetes)、海植戴尔门(Hypochytridiomycetes)、康曼门(Commation)、瑞可罗门(Reticulosphaera)、普莱格门(Pelagomonas)、普莱格可门(Pelagococcus)、欧里可拉门(Ollicola)、奥瑞可门(Aureococcus)、小壳藻门(Parmales)、硅藻门(Diatoms)、黄藻门(Xanthophytes)、褐藻门(Phaeophytes,褐藻)、黄绿藻门(Eustigmatophytes)、绿鞭藻门(Raphidophytes)、新纽门(Synurids)、爱科斯汀门(Axodines)(包括根单鞭金藻目(Rhizochromulinales)、柄钟藻目(Pedinellales)、硅鞭藻目(Dictyochales))、克里索达纲(Chrysomeridales)、萨西诺达纲(Sarcinochrysidales)、水树藻目(Hydrurales)、蛰居金藻目(Hibberdiales)和色金藻目(Chromulinales)。微藻类的其他优选组包括绿藻和腰鞭毛虫的成员,包括Crypthecodiurn属的成员。
所述植物细胞可以特别地选自十字花科(Brassicaceae)、胡颓子科(Elaeagnaceae)和豆科(Fabaceae)的细胞。十字花科的细胞可以选自芸苔(Brassica)属,特别是选自油菜籽、芜菁油菜和印度芥菜。胡颓子科的细胞可以选自胡颓子(Elaeagnus)属,特别是选自油橄榄(Oleae europaea);豆科的细胞可以选自大豆属(genus Glycine),特别是选自大豆(Glycine max)物种。
在一些实施方式中,油中的ω-3多不饱和脂肪酸的总量为每1克油至少约400mg。
在其他实施方式中,油中的ω-3多不饱和脂肪酸的总量为每1克油至少约500mg。
在另外其他实施方式中,油中的ω-3多不饱和脂肪酸的总量为每1克油约400mg至约800mg。
在一些实施方式中,该油包含每1克油约100mg至约300mg EPA和每1克油约200mg至约500mg DHA。
在另一个实施方式中,该油包含每1克油约100mg至约250mg EPA;每1克油约250mg至约400mg DHA。
在一些实施方式中,该油按ω-3多不饱和脂肪酸的总重量计包含1:1至1:30或1:1至1:5的EPA:DHA比率。
在一些实施方式中,该油按ω-3多不饱和脂肪酸的总重量计包含1:1至1:4的EPA:DHA比率。
在一些实施方式中,该油按ω-3多不饱和脂肪酸的总重量计包含至少1:1、至少1:2、至少1:3或至少1:4的EPA:DHA比率。
在一些实施方式中,该油是包含至少约25重量%的二十二碳六烯酸,至少约10重量%的二十碳五烯酸,小于2重量%、优选小于1重量%的水分含量和在水分中小于5重量%游离脂肪酸的微生物油。
本发明还涉及包含至少约10重量%的三酰基甘油级分的微生物油,其中所述三酰基甘油级分中的至少约12重量%的脂肪酸是二十碳五烯酸,其中所述三酰基甘油级分中的至少约25重量%的脂肪酸是二十二碳六烯酸,且其中所述三酰基甘油级分中的小于约5重量%的脂肪酸是花生四烯酸。
在一些实施方式中,微生物油按重量计包含约0%、至少约0.1%、至少约0.2%、至少约0.5%、至少约1%、至少约1.5%、至少约2%,或至少约5%的固醇酯级分。在一些实施方式中,微生物油按重量计包含约0%至约1.5%、约0%至约2%、约0%至约5%、约1%至约1.5%、约0.2%至约1.5%、约0.2%至约2%,或约0.2%至约5%的固醇酯级分。在一些实施方式中,微生物油按重量计包含约5%以下、约4%以下、约3%以下、约2%以下、约1%以下、约0.5%以下、约0.3%以下、约0.2%以下、约0.5%以下、约0.4%以下、约0.3%以下,或约0.2%以下的固醇酯级分。
在一些实施方式中,微生物油按重量计包含至少约35%、至少约40%、至少约45%、至少约50%、至少约55%、至少约60%、至少约65%、至少约70%、至少约75%、至少约80%、至少约85%,或至少约90%的三酰基甘油级分。在一些实施方式中,微生物油按重量计包含约35%至约98%、约35%至约90%、约35%至约80%、约35%至约70%、约35%至约70%、约35%至约65%、约40%至约70%、约40%至约65%、约40%至约55%、约40%至约50%、约65%至约95%、约75%至约95%、约75%至约98%、约80%至约95%、约80%至约98%、约90%至约96%、约90%至约97%、约90%至约98%、约90%、约95%、约97%,或约98%的三酰基甘油级分。
在一些实施方式中,微生物油按重量计包含至少约10%、至少约11%、至少约12%、至少约13%、至少约14%、至少约15%、至少约16%、至少约17%、至少约18%、至少约19%,或至少约20%的二酰基甘油级分。在一些实施方式中,微生物油按重量计包含约10%至约45%、约10%至约40%、约10%至约35%、约10%至约30%、约15%至约40%、约15%至约35%,或约15%至约30%的二酰基甘油级分。在一些实施方式中,微生物油按重量计包含至少约0.2%、至少约0.3%、至少约0.4%、至少约0.5%、至少约1%、至少约5%、至少约10%、至少约11%、至少约12%、至少约13%、至少约14%、至少约15%、至少约16%、至少约17%、至少约18%、至少约19%,或至少约20%的1,2-二酰基甘油级分。在一些实施方式中,微生物油按重量计包含约0.2%至约45%、约0.2%至约30%、约0.2%至约20%、约0.2%至约10%、约0.2%至约5%、约0.2%至约1%、约0.2%至约0.8%、约0.4%至约45%、约0.4%至约30%、约0.4%至约20%、约0.4%至约10%、约0.4%至约5%、约0.4%至约1%、约0.4%至约0.8%、约0.5%至约1%、约0.5%至约0.8%、约10%至约45%、约10%至约40%、约10%至约35%、约10%至约30%、约15%至约40%、约15%至约35%、约15%至约30%,或约15%至约25%的二酰基甘油级分。在一些实施方式中,微生物油按重量计包含至少约0.1%、至少约0.2%、至少约0.5%、至少约1%、至少约2%、至少约2.5%,或至少约3%的1,3-二酰基甘油级分。在一些实施方式中,微生物油按重量计包含至少约0.3%、至少约0.4%、至少约0.5%、至少约1%、至少约1.5%、至少约2%,或至少约5%的固醇级分。
在一些实施方式中,微生物油按重量计包含约0.3%至约5%、约0.3%至约2%、约0.3%至约1.5%、约0.5%至约1.5%、约1%至约1.5%、约0.5%至约2%、约0.5%至约5%、约1%至约2%,或约1%至约5%的固醇级分。在一些实施方式中,微生物油按重量计包含约5%以下、约4%以下、约3%以下、约2%以下、约1.5%以下,或约1%以下的固醇级分。
在一些实施方式中,微生物油按重量计包含至少约2%、至少约5%,或至少约8%的磷脂级分。在一些实施方式中,微生物油按重量计包含约2%至约25%、约2%至约20%、约2%至约15%、约2%至约10%、约5%至约25%、约5%至约20%、约5%至约20%、约5%至约10%,或约7%至约9%的磷脂级分。在一些实施方式中,微生物油按重量计包含小于约20%、小于约15%、小于约10%、小于约9%,或小于约8%的磷脂级分。在一些实施方式中,微生物油基本上不含磷脂。在一些实施方式中,微生物油按重量计包含小于约2%、小于约1.5%、小于约1%,或小于约0.5%油的不皂化物。微生物油中存在的脂质类别,例如三酰基甘油级分,可以通过快速色谱法分离,并通过薄层色谱法(TLC)分析,或通过本领域已知的其他方法分离和分析。
在一些实施方式中,选自三酰基甘油级分、游离脂肪酸级分、固醇级分、二酰基甘油级分及它们的组合的微生物油和/或其一种或多种级分按重量计包含至少约5%、至少约10%、大于约10%、至少约12%、至少约13%、至少约14%、至少约15%、至少约16%、至少约17%、至少约18%、至少约19%、至少约20%、至少约25%、至少约30%、至少约35%、至少约40%,或至少约45%EPA。在一些实施方式中,选自三酰基甘油级分、游离脂肪酸级分、固醇级分、二酰基甘油级分及它们的组合的微生物油和/或其一种或多种级分按重量计包含约5%至约55%、约5%至约50%、约5%至约45%、约5%至约40%、约5%至约35%、约5%至约30%、约10%至约55%、约10%至约50%、约10%至约45%、约10%至约40%、约10%至约35%、约10%至约30%、至少约12%至约55%、至少约12%至约50%、至少约12%至约45%、至少约12%至约40%、至少约12%至约35%,或至少约12%至约30%、约15%至约55%、约15%至约50%、约15%至约45%、约15%至约40%、约15%至约35%、约15%至约30%、约15%至约25%、约15%至约20%、约20%至约55%、约20%至约50%、约20%至约45%、约20%至约40%,或约20%至约30%的EPA。在一些实施方式中,选自三酰基甘油级分、二酰基甘油级分、固醇级分、固醇酯级分、游离脂肪酸级分、磷脂级分及它们的组合的微生物油和/或其一种或多种级分按重量计包含至少约5%、至少约10%、至少约15%、至少约20%、至少约25%、至少约30%、至少约35%、至少约40%、至少约50%,或至少约60%DHA。在一些实施方式中,选自三酰基甘油级分、二酰基甘油级分、固醇级分、固醇酯级分、游离脂肪酸级分、磷脂级分,及它们的组合的微生物油和/或其一种或多种级分按重量计包含约5%至约60%、约5%至约55%、约5%至约50%、约5%至约40%、约10%至约60%、约10%至约50%、约10%至约40%、约20%至约60%、约25%至约60%、约25%至约50%、约25%至约45%、约30%至约50%、约35%至约50%,或约30%至约40%的DHA。在一些实施方式中,选自三酰基甘油级分、二酰基甘油级分、固醇级分、固醇酯级分、游离脂肪酸级分、磷脂级分及它们的组合的微生物油和/或其一种或多种级分按重量计包含约10%以下、约9%以下、约8%以下、约7%以下、约6%以下、约5%以下、约4%以下、约3%以下、约2%以下,或约1%以下的DHA。在一些实施方式中,选自三酰基甘油级分、二酰基甘油级分、固醇级分、固醇酯级分、游离脂肪酸级分、磷脂级分及它们的组合的微生物油和/或其一种或多种级分按重量计包含约1%至约10%、约1%至约5%、约2%至约5%、约3%至约5%,或约3%至约10%的DHA作为脂肪酸。在一些实施方式中,选自三酰基甘油级分、二酰基甘油级分、固醇级分、固醇酯级分、游离脂肪酸级分、磷脂级分、及它们的组合的微生物油和/或其一种或多种级分,基本上不含DHA。在一些实施方式中,选自三酰基甘油级分、二酰基甘油级分、固醇级分、固醇酯级分、游离脂肪酸级分、磷脂级分及它们的组合的微生物油和/或其一种或多种级分按重量计包含约0.1%至约5%、约0.1%至小于约5%、约0.1%至约4%、约0.1%至约3%、约0.1%至约2%、约0.2%至约5%、约0.2%至小于约5%、约0.2%至约4%、约0.2%至约3%、约0.2%至约2%、约0.3%至约2%、约0.1%至约0.5%、约0.2%至约0.5%、约0.1%至约0.4%、约0.2%至约0.4%、约0.5%至约2%、约1%至约2%、约0.5%至约1.5%,或约1%至约1.5%的ARA。在一些实施方式中,选自三酰基甘油级分、二酰基甘油级分、固醇级分、固醇酯级分、游离脂肪酸级分、磷脂级分及它们的组合的微生物油和/或其一种或多种级分按重量计包含约5%以下、小于约5%、约4%以下、约3%以下、约2%以下、约1.5%以下、约1%以下、约0.5%以下、约0.4%以下、约0.3%以下、约0.2%以下,或约0.1%以下的ARA。在一些实施方式中,选自三酰基甘油级分、二酰基甘油级分、固醇级分、固醇酯级分、游离脂肪酸级分、磷脂级分及它们的组合的微生物油和/或其一种或多种级分,基本上不含ARA。在一些实施方式中,选自三酰基甘油级分、二酰基甘油级分、固醇级分、固醇酯级分、游离脂肪酸级分、磷脂级分及它们的组合的微生物油和/或其一种或多种级分按重量计包含约0.4%至约2%、约0.4%至约3%、约0.4%至约4%、约0.4%至约5%、约0.4%至小于约5%、约0.5%至约1%、约0.5%至约2%、约0.5%至约3%、约0.5%至约4%、约0.5%至约5%、约0.5%至小于约5%、约1%至约2%、约1%至约3%、约1%至约4%、约1%至约5%,或约1%至小于约5%的DPA n-6。在一些实施方式中,选自三酰基甘油级分、二酰基甘油级分、固醇级分、固醇酯级分、游离脂肪酸级分、磷脂级分及它们的组合的微生物油和/或其一种或多种级分按重量计包含约5%、小于约5%、约4%以下、约3%以下、约2%以下、约1%以下、约0.75%以下、约0.6%以下,或约0.5%以下的DPA n-6。在一些实施方式中,选自三酰基甘油级分、二酰基甘油级分、固醇级分、固醇酯级分、游离脂肪酸级分、磷脂级分及它们的组合的微生物油和/或其一种或多种级分,基本上不含DPA n-6。在一些实施方式中,选自三酰基甘油级分、二酰基甘油级分、固醇级分、固醇酯级分、游离脂肪酸级分、磷脂级分及它们的组合的微生物油和/或其一种或多种级分按重量计包含具有约5%以下、小于约5%、约4%以下、约3%以下,或约2%以下的油酸(18:1n-9)、亚油酸(18:2n-6)、亚麻酸(18:3n-3)、二十碳烯酸(20:1n-9)、芥酸(22:1n-9)、十八碳四烯酸(18:4n-3)或其组合的脂肪酸。
在另一个实施方式中,微生物油包含ω-3多不饱和脂肪酸,其包含占ω-3多不饱和脂肪酸总量的约>90重量%的量的DHA和EPA,其中EPA的量为DHA和EPA的总量的约10重量%至约60重量%,且DHA的量为DHA和EPA的总量的约40重量%至约90重量%。
本发明的油组合物包括但不限于饲料和食品、药物组合物和化妆品。
在一些实施方式中,油组合物是动物饲料添加剂。“动物”包括属于动物界的非人类生物,且包括但不限于水生动物和陆生动物。术语“动物饲料”或“动物食物”是指旨在用于非人类动物的任何食物,无论是鱼;商业鱼;观赏鱼;鱼苗;双壳类;软体动物;甲壳动物;贝类;虾;幼虾;卤虫(artemia);轮虫;盐水虾;滤食动物;两栖动物;爬行动物;哺乳动物;家畜;耕畜;动物园动物;运动类动物;种畜;比赛类动物;表演类动物;活化石动物(heirloomanimals);稀有或濒危动物;伴侣动物;宠物如狗、猫、豚鼠、兔、大鼠、小鼠或马;灵长类动物如猴(例如,卷尾猴、恒河猴、非洲绿猴、赤猴、食蟹猴和长尾猴)、猿、猩猩、狒狒、长臂猿和黑猩猩;犬科动物如狗和狼;猫科动物如猫、狮子和虎;马科动物如马、驴和斑马;食用动物如奶牛、牛、猪和绵羊;有蹄动物如鹿和长颈鹿;或啮齿动物如小鼠、大鼠、仓鼠和豚鼠;等等。动物饲料包括但不限于水产养殖饲料、家畜饲料,包括宠物饲料、动物园动物饲料、役畜饲料、畜牲饲料及其组合。
在动物饲料添加剂的一个实施方式中,ω-3多不饱和脂肪酸的总量为每1克油至少约400mg,优选为每1克油约500mg,其中油为微生物油。
在动物饲料添加剂的一些实施方式中,微生物油包含每1克油约100mg至约300mgEPA和每1克油约200mg至约500mg DHA。在另一个实施方式中,微生物油按ω-3多不饱和脂肪酸的总重量计包含1:1至1:30或1:1至1:5的EPA:DHA的比率。
根据本发明的微生物油衍生自微生物生物质,所述微生物生物质包含细胞,且优选基本上由以下分类单元的这种细胞组成:网粘菌纲(Labyrinthulomycetes)(网粘菌(Labyrinthulea)、网粘真菌(net slime fungi)、网粘菌(slime nets)),特别是破囊壶菌科(Thraustochytriaceae)的那些。破囊壶菌科(Thraustochytrids)包括以下属:Althomia、不动茶菌属(Aplanochytrium)、Aurantiochytrium、Botryochytrium、Elnia、日本壶菌属(Japonochytrium)、Oblongichytrium、Parietichytrium、裂殖壶菌属(Schizochytrium)、Sicyoidochytrium、破囊壶菌属(Thraustochytrium),和吾肯氏壶菌属(Ulkenia)。生物质特别优选包含以下属的细胞:Aurantiochytrium、Oblongichytrium、裂殖壶菌属,或破囊壶菌属,尤其是裂殖壶菌属。
在本发明的一个非常优选的实施方式中,采用同时产生大量EPA和DHA的细胞,特别是裂殖壶菌菌株,其中DHA优选以至少20重量%的量,优选以至少30重量%的量,特别是以30至50重量%的量产生,且EPA以至少5重量%的量,优选以至少10重量%的量,特别是以10至20重量%的量(分别相对于细胞中包含的脂质的总量)产生。产生DHA和EPA的裂殖壶菌菌株可通过连续诱变,随后适当选择表现出优异的EPA和DHA产生和特定的EPA:DHA比率的突变菌株来获得。能够引起酵母细胞遗传改变的任何化学或非化学(例如紫外线(UV)辐射)试剂都可以用作诱变剂。这些试剂可以单独使用或彼此结合使用,且化学试剂可以纯净地或与溶剂一起使用。
如前所述,可同时产生大量EPA和DHA的裂殖壶菌属的微生物的优选物种根据ATCC登记号PTA-10208、PTA-10209、PTA-10210,或PTA-10211、PTA-10212、PTA-10213、PTA-10214、PTA-10215保藏。
根据本发明的微生物油可以根据下述方法a制造。
制造过程
从微生物生物质残渣分离含多不饱和脂肪酸(PUFA)的油的有效方法包括以下步骤:
a)提供包含细胞的生物质的悬浮液,所述细胞包含含PUFA的脂质;
b)裂解所述生物质的细胞;
c)将如步骤(b)中获得的悬浮液加热至80℃至100℃,优选85℃至95℃,更优选约90℃的温度,同时将pH调节至9.5至11.5,优选10.0至11.0,更优选10.3至10.7的值;
d)将温度和pH值保持在如(c)中描述的范围至少10小时,优选15至40小时,更优选20至36小时。
e)中和并将油与生物质分离。
步骤(c)和(d)导致如通过裂解生物质的细胞而获得的含油的轻相与含水、细胞碎片、盐和残留油的重相分离。在本申请的上下文中,轻相和重相的这种分离也称为“反乳化(de-emulsification)”或“反乳化(demulsification)”。
步骤(d)中的措施的顺序并不重要。可以在调节pH值之前或之后进行温度调节。
优选地,在该方法的步骤(b)、(c)和(d)中,通过使用搅拌器和/或搅动器将悬浮液连续混合。在方法步骤(c)和/或(d)中,优选地,特别是如在WO 2015/095694中所公开的,应用低剪切搅动和/或轴向流搅动。适用于在步骤(c)和/或(d)之前和期间搅动的叶轮特别地包括直叶片叶轮、Rushton叶片叶轮、轴向流叶轮、径向流叶轮、凹叶片圆盘叶轮、高效叶轮、螺旋桨、桨叶、涡轮及它们的组合。
生物质的细胞的裂解可以通过本领域技术人员已知的方法进行,特别是通过酶、机械、物理或化学,或通过它们的组合进行。
取决于暴露的时间和/或施加的力的程度,可以获得仅包含裂解细胞的组合物或包含细胞碎片和完整细胞的混合物的组合物。就此而言,术语“含有裂解脂质的生物质”是指一种悬浮液,其含有被生物质的细胞释放的水、细胞碎片和油,但除此之外还可以包含其他组分,特别是盐、完整细胞、裂解的细胞的其他内含物以及发酵培养基的组分,特别是营养成分。在本发明的一个优选的实施方式中,仅少量的完整细胞,特别是小于20%,优选小于10%,更优选小于5%(相对于如在生物质的细胞裂解之前存在的完整细胞的总数)在裂解细胞的步骤之后,存在于裂解的生物质中。
细胞的裂解可以例如通过利用法国细胞压机、超声仪、均质器、微流化器、球磨机、棒磨机、砾磨机、珠磨机、高压磨辊、垂直轴冲击器、工业共混器、高剪切混合器、桨式混合器和/或polytron均质器来实现。
在一个优选的实施方式中,细胞裂解包括通过应用细胞壁降解酶对细胞进行酶处理。细胞壁降解酶优选选自蛋白酶、纤维素酶(例如Cellustar CL(Dyadic)、FibrezymeG2000(Dyadic)、Celluclast(Novozymes)、Fungamyl(Novozymes)、Viscozyme L(Novozymes))、半纤维素酶、几丁质酶、果胶酶(例如Pectinex(Novozymes))、蔗糖酶、麦芽糖酶、乳糖酶、α-葡萄糖苷酶、β-葡萄糖苷酶、淀粉酶(例如Alphastar Plus(Dyadic);Termamyl(Novozymes))、溶菌酶、神经氨酸酶、半乳糖苷酶、α-甘露糖苷酶、葡糖醛酸酶、透明质酸酶、支链淀粉酶、葡糖脑苷脂酶、半乳糖神经酰胺酶、乙酰基半乳糖苷酶(acetylgalactosaminidases)、岩藻糖苷酶、己糖胺酶、艾杜糖苷酸酶、麦芽糖酶-葡糖淀粉酶、木聚糖酶(例如Xylanase Plus(Dyadic)、Pentopan(Novozymes))、β-葡聚糖酶(例如Vinoflow Max(Novozymes)、Brewzyme LP(Dyadic))、甘露聚糖酶及它们的组合。蛋白酶可以选自丝氨酸蛋白酶、苏氨酸蛋白酶、半胱氨酸蛋白酶、天冬氨酸蛋白酶、金属蛋白酶、谷氨酸蛋白酶、碱性蛋白酶(枯草杆菌蛋白酶)及它们的组合。几丁质酶可以是壳三糖酶。果胶酶可以选自果胶降解酶(pectolyases)、果胶裂解酶(pectozymes)、多聚半乳糖醛酸酶及它们的组合。
酶优选作为浓缩酶溶液,优选以相对于浓缩酶溶液的量0.01至1.5重量%的量,更优选以0.03至1.0重量%的量,特别是以0.05至0.5重量%的量加入,所述浓缩酶溶液的量相对于在加入浓缩酶溶液后悬浮液的总量加入。
在该方法的另一个优选实施方式中,在裂解生物质的细胞之后且在反乳化步骤之前,将悬浮液浓缩至30至60重量%,更优选35至55重量%,特别是40至50重量%的总干物质含量。
悬浮液的浓缩优选通过在不高于100℃,优选70℃至100℃,更优选80℃至90℃的温度下蒸发水直至30至60重量%,更优选35至55重量%,特别是40至50重量%的总干物质含量来进行。
悬浮液的浓缩优选在强制循环蒸发器(例如可从德国GEA获得)中进行以允许快速除去水。
通常,根据本发明,可以通过使用本领域技术人员已知的碱或酸来调节pH值。降低pH可以特别地通过使用有机或无机酸如硫酸、硝酸、磷酸、硼酸、盐酸、氢溴酸、高氯酸、次氯酸、亚氯酸、氟硫酸、六氟磷酸、乙酸、柠檬酸、甲酸或其组合来进行。由于期望避免高含量的氯化物,在本发明的一个优选实施方式中,在本发明的方法中不使用或仅使用少量的盐酸。根据本发明,硫酸是降低pH值的优选物质。–提高pH可以特别地通过使用有机或无机碱如氢氧化物(特别是氢氧化钠、氢氧化锂、氢氧化钾和/或氢氧化钙)、碳酸盐(特别是碳酸钠、碳酸钾或碳酸镁)和/或碳酸氢盐(特别是碳酸氢锂、碳酸氢钠和/或碳酸氢钾)来进行。–由于易于处理,酸和碱优选以液体形式,特别是作为浓缩液使用。因此,苛性钠是用于增加pH值的优选物质。
该方法优选地包括作为进一步的步骤,从如步骤(d)中获得的反乳化组合物收获含PUFA的脂质。
含PUFA的脂质的收获优选包括中和反乳化的悬浮液且然后将由此获得的含油的轻相与含水、盐、细胞碎片和残留油的重相分离。
反乳化的组合物的中和优选通过加入酸,优选硫酸来调节5.5至8.5,特别是6.5至8.5,优选7.0至8.0的pH值来实现。在将轻相与重相开始分离之前,可以将由此获得的中和的组合物在所述pH值下搅拌几分钟直至几小时。
含油的轻相与含水、盐和细胞碎片的重相分离优选通过机械手段且优选在60-90℃,更优选70-80℃的温度,且在优选6-9,更优选7-8.5的pH值下实现。“机械手段”特别是指本领域技术人员已知的过滤和离心方法。
在分离含油的轻相后,可以通过应用本领域技术人员已知的方法,特别是精制、漂白、除臭和/或防冻来进一步处理由此获得的含PUFA的油。
该方法的一个特别的优点是,通过在分离之前将pH调节至小于8.5,优选小于7.5,可以减缓游离脂肪酸到油的水相(水分),从而使油中的残留水分包含小于约8重量%,优选小于约5重量%的游离脂肪酸。
本发明的方法允许将生物质中包含的油与发酵液中包含的细胞碎片和其他物质非常有效地分离。通过使用本发明的方法,可以从生物质分离优选地大于80重量%,特别是大于90重量%的生物质中所含的油并分离出来。
结果是通过应用本发明的方法获得的油相对于迄今为止在现有技术中公开的含PUFA的油具有一些有利的特征。特别地,它表现出非常低的氧化值,低含量的游离脂肪酸和杂质,非常低的粘度和非常高的闪点。
游离脂肪酸的含量根据AOCS官方方法AOCS Ca 5a-40确定。水分含量根据AOCS官方方法AOAC 930.15,935.29确定。不溶杂质的含量根据AOCS官方方法AOCS 3a-46确定。DHA和EPA的量根据AOCS官方方法AOCS Ce 1b-89确定。总脂肪量根据AOCS官方方法AOCS996.06确定。粗脂肪的量根据AOCS官方方法AOAC 920.39,954.02确定。
实施例
实施例1:微生物油的产生
在搅动的容器中,将生物质密度的超过100g/l的含有微生物细胞(Schizochytrium sp.)的未洗涤的细胞肉汤加热至60℃。在加热悬浮液后,在以0.5重量%(按肉汤重量计)的量的液体形式加入碱性蛋白酶(2.4FG(Novozymes))之前,通过使用苛性钠(50重量%的NaOH溶液)将pH调节至7.5。在60℃下继续搅拌3小时。之后,将裂解的细胞混合物转移至强制循环蒸发器(从德国GEA获得)中并加热至85℃的温度。将混合物在强制循环蒸发器中浓缩,直到达到约30重量%的总干物质含量。将浓缩的裂解细胞混合物转移至新容器中,在低剪切搅动下加热至90℃,同时通过加入苛性钠将pH调节至10.5。继续低剪切搅动约30小时,同时通过加入苛性钠将温度保持在90℃且pH保持高于9.0。
之后,通过加入硫酸来调节7.5的pH将所得的反乳化混合物中和。通过使用圆盘堆叠分离器(Alfa Laval圆盘堆叠离心机,LAPX 404/Clara 20)机械地将相分离成含油的轻相和含水、细胞碎片、残留油和盐的重相。
由于有效的反乳化,可以在不加入有机溶剂或氯化钠的情况下从生物质分离超过90重量%的油。且最后,该油有效地稳定且不显示胶凝性质。它在油的残留水分中包含小于5重量%的游离脂肪酸。
实施例2–作为动物饲料添加剂的微生物油
下面显示用作水产养殖中的动物饲料添加剂的根据本发明的微生物原油的说明。
DHA+EPA,mg/g油 | 最小500mg/g |
DHA含量,mg/g油 | 最小250mg/g(mind 25%->40%) |
EPA含量,mg/g油 | 最小100mg/g(mind 10%->25%) |
比率EPA:DHA最小 | 1:04 |
比率EPA:DHA最大 | 1:01 |
游离脂肪酸 | 最大5% |
水分 | 最大0.75% |
DPA n-3 | <6 |
花生四烯酸,% | <2 |
硬脂酸,% | <2.5 |
棕榈酸,% | <30 |
粗脂肪 | >92% |
Claims (15)
1.一种油,其包含至少约25重量%的ω-3多不饱和脂肪酸和如下的水分含量,所述水分含量含有小于约8重%,优选小于约5重量%的游离脂肪酸。
2.根据权利要求1所述的油,其包含小于2重量%,优选小于1重量%的水分含量。
3.根据权利要求1或2所述的油,其包含至少约25重量%的二十二碳六烯酸。
4.根据权利要求3所述的油,其还包含至少约10重量%的二十碳五烯酸。
5.根据权利要求1至4中任一项所述的油,其中所述油中的ω-3多不饱和脂肪酸的总量为每1克油至少约400mg。
6.根据权利要求1至4中任一项所述的油,其中所述油中的ω-3多不饱和脂肪酸的总量为每1克油至少约500mg。
7.根据权利要求1至4中任一项所述的油,其中所述油包含每1克油约100mg至约250mgEPA和每1克油约250mg至约400mg DHA。
8.根据权利要求4至7中任一项所述的油,其中所述油按ω-3多不饱和脂肪酸的总重量计包含1:1至1:30或1:1至1:5的EPA:DHA比率。
9.根据权利要求1至4中任一项所述的油,其中所述油是植物油。
10.根据权利要求1至4中任一项所述的油,其中所述油是微生物油。
11.根据权利要求10所述的油,其是由裂殖壶菌属(Schizochytrium sp)产生的。
12.根据权利要求10或11所述的微生物油,其包含至少约25重量%的二十二碳六烯酸,至少约10重量%的二十碳五烯酸,小于2重量%、优选小于1重量%的水分含量,以及在所述水分中小于5重量%的游离脂肪酸。
13.根据权利要求10至12中任一项所述的微生物油,其中所述ω-3多不饱和脂肪酸的总量为每1克油约400mg至约800mg。
14.根据权利要求10至13中任一项所述的微生物油,其中所述油按ω-3多不饱和脂肪酸的总重量计包含选自至少1:1、至少1:2、至少1:3或至少1:4的EPA:DHA比率。
15.根据权利要求10至12中任一项所述的微生物油,其包含至少约10重量%的三酰基甘油级分,其中所述三酰基甘油级分中的至少约12重量%的脂肪酸是二十碳五烯酸,其中所述三酰基甘油级分中的至少约25重量%的脂肪酸是二十二碳六烯酸,并且其中所述三酰基甘油级分中的小于约5重量%的脂肪酸是花生四烯酸。
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BR112019000435A2 (pt) | 2016-07-13 | 2019-04-30 | Evonik Degussa Gmbh | método para separar lipídios de uma biomassa contendo lipídios lisados |
US11352651B2 (en) | 2016-12-27 | 2022-06-07 | Evonik Operations Gmbh | Method of isolating lipids from a lipids containing biomass |
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