CN107405537A - 从发酵液分离鼠李糖脂的方法 - Google Patents

从发酵液分离鼠李糖脂的方法 Download PDF

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CN107405537A
CN107405537A CN201680009494.7A CN201680009494A CN107405537A CN 107405537 A CN107405537 A CN 107405537A CN 201680009494 A CN201680009494 A CN 201680009494A CN 107405537 A CN107405537 A CN 107405537A
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R·J·马里奥特
P·S·史蒂文森
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Abstract

一种从鼠李糖脂发酵混合物提取至少一种鼠李糖脂化合物的方法,所述方法包括以下步骤:(a)将惰性吸附载体与鼠李糖脂发酵混合物混合;(b)在高于31℃的温度和高于73巴的压力下将混合物溶解在有机溶剂中;(c)分离至少一种脂肪化合物;(d)向所述有机溶剂中加入共溶剂;(e)改变温度和/或压力;(f)分离第一鼠李糖脂化合物;和(g)分离第二鼠李糖脂化合物。

Description

从发酵液分离鼠李糖脂的方法
技术领域
本发明涉及从鼠李糖脂发酵培养基分离鼠李糖脂化合物。
背景技术
生物表面活性剂如鼠李糖脂可以通过使用可再生生物质和各种细菌如铜绿假单胞菌或其他合适的微生物生物体与可以是植物油或动物油或糖的原料一起发酵而生产。
然而,必须进一步处理所得发酵混合物或“发酵液”以除去/纯化鼠李糖脂。其他发酵液成分包括不完全反应的进料底物,例如甘油三酯、甘油二酯和甘油单酯,还有脂肪酸,以及细胞成分和糖等。高回收和纯化成本是鼠李糖脂的大规模商业化所要克服的主要障碍。
Heyd等(Anal.Bioanal.Chem.(2008)391,1579)提供了生物表面活性剂分析和纯化的总结。Reiling等(Appl.Environ.Microbiol.(1986),51(5),985)公开了使用铜绿假单胞菌生产鼠李糖脂生物表面活性剂的中试方法。EP 0317036公开了一种制备L-鼠李糖的方法,该方法包括在低pH下在有机羧酸的水溶液中加热。
发明内容
本发明的目的是提供从发酵混合物提取鼠李糖脂化合物的改进方法。
因此,本发明提供了一种从鼠李糖脂混合物提取至少一种鼠李糖脂化合物的方法,所述方法包括以下步骤:
(a)将惰性吸附载体与鼠李糖脂混合物混合;
(b)在高于31℃的温度和高于73巴的压力下将混合物溶解在有机溶剂中;
(c)分离至少一种脂肪化合物;
(d)向所述有机溶剂中加入共溶剂;
(e)改变温度和/或压力;
(f)分离第一鼠李糖脂化合物;和
(g)分离第二鼠李糖脂化合物。
采用本发明,用于收获鼠李糖脂化合物的方法在效力、准确性和环境影响方面大大改善。
优选地,步骤(f)和(g)的鼠李糖脂提取是在共溶剂的加入(d)和步骤(e)中温度和/或压力的增加之后。
优选地,步骤(c)在步骤(d)至(f)之前。优选地,水也在步骤(c)中除去。
优选地,步骤(c)采用并联的多个提取/分离容器进行,借此水在一个容器中分离而脂肪化合物在不同的容器中分离。
优选地,脱脂步骤(c)被延长至几乎没有油或水被提取。
优选地,步骤是按字母顺序排序的。
优选地,在步骤(e)中减小压力。优选地,这足以提供有机溶剂中的相变。在CO2的情况下,此压力减小应足以将超临界CO2转化为液体CO2
优选地,多个提取/分离器容器被用于步骤(f)和(g)。
一般而言,混合和溶解步骤(a)和(b)在步骤(c)至(g)之前。步骤(c)至(g)可以以任何适当的顺序进行。例如,加入共溶剂的步骤(d)优选在步骤(e)中改变温度和/或压力之前。在一些实例中,分离脂肪化合物步骤(c)可以在步骤(d)和(e)之后。
在步骤(e)中,可以增加或减小温度,并且维持压力。或者,可以维持温度并增加或减小压力。或者,可以增加温度和压力,或者可以减小温度和压力。或者,可以增加温度和压力中的一个,并且可以减小温度和压力中的另一个。
优选地,在步骤(e)中增加温度和压力。或者,优选地,在步骤(e)中维持温度并降低压力。
优选地,第一鼠李糖脂化合物包含单鼠李糖脂(通常称为“R1”)。
优选地,第二鼠李糖脂化合物包含二鼠李糖脂(通常称为“R2”)。
如本文所用,术语“鼠李糖脂混合物”是指鼠李糖脂、脂肪酸和甘油酯(包括甘油单酯、甘油三酯、甘油二酯)和糖、蛋白质、残留细胞/细胞材料的微生物发酵后混合物。这可以包含鼠李糖脂发酵液或在其它处理后的鼠李糖脂发酵液的粗产物或富鼠李糖脂相。
如本文所用,术语“惰性吸附载体”可以包含单一结构或结构阵列。术语“惰性吸附剂”可以替代地与如本文所用的术语“惰性吸附载体”互换使用。
优选地,惰性吸附载体包含大表面积并具有多孔结构(高孔隙度)。所述孔隙度可通过载体结构(例如包含多个孔的一个或多个主体)和/或通过结构阵列的构造(例如自由流动的粉末形式的珠或球或颗粒的阵列,其中所述颗粒自身也可以包含孔)固有地提供。
优选地,惰性吸附载体包含硅藻土。
优选地,惰性吸附载体包含Celite。优选地,Celite可包括 其全部由Celite Corp.生产。
优选地,惰性吸附载体材料具有直径范围为7.5至30nm的孔。优选地,孔隙度范围为1至4cm3/g。优选地,比表面积范围在5至250m2/g之间。优选地,在颗粒状惰性吸附载体材料的情况下,平均颗粒直径是在1μm至1.5mm的范围内。
在自由流动的颗粒状惰性吸附的情况下,基于负载粉末的重量,混合物在载体上的负载量优选在5重量%和60重量%之间。优选地,其范围在20重量%和55重量%之间,更优选地,在30重量%和50重量%之间。
鼠李糖脂混合物优选以水性形式吸附在惰性吸附载体上。
优选地,有机溶剂是二氧化碳(CO2)。借助于步骤(b)和(e)的二氧化碳处于超临界状态。优选地,其对于(b)和(e)二者均处于超临界状态。
对于使用处于超临界状态的二氧化碳的分离,在高于31℃的温度和高于73巴的压力下进行分离反应。优选地,温度范围在31℃和200℃之间,更优选在31℃和80℃之间。优选地,压力范围在73巴和1100巴之间,更优选在200巴和700巴之间。考虑除二氧化碳以外的溶剂。在一些情况下,步骤(b)可在其他温度和压力下发生。在该情况下,步骤(b)可以包括将混合物溶解在有机溶剂中。
最优选地,步骤(a)至(d)在450巴的压力和40℃的温度下进行,且在步骤(e)中,通过降低至300巴而改变压力(温度保持在40℃),使得步骤(f)和(g)相对于步骤(a)至(d)在减压下进行。
共溶剂优选包含醇。共溶剂优选包含乙醇或甲醇。共溶剂最优选包含乙醇(例如IMS:工业甲基化酒精)。
优选地,增加所用共溶剂的量以除去不同的鼠李糖脂化合物。共溶剂的量优选从(总溶剂重量的)2.5重量%增加至10重量%以逐步除去单鼠李糖脂和二鼠李糖脂。
优选地,用于除去单鼠李糖脂,所用共溶剂的量为2.5重量%。优选地,用于除去二鼠李糖脂,所用共溶剂的量为10重量%。优选地,用于除去单鼠李糖脂和二鼠李糖脂的混合物,所用共溶剂的量为5重量%。
实施例
使用具有图1所示的质谱鉴定的组成的粗鼠李糖脂生物质的提取试验。以30mg/L在甲醇中制备溶液后进行质谱分析。采用电喷雾离子化(ESI)在负离子化模式下进行分析,扫描范围为100-1200amu,流速为20μL/min。在图1中,我们可以看到不同的化合物:脂肪酸和甘油酯、单鼠李糖(R1)和二鼠李糖(R2)。
以3.7份载体与1份鼠李糖脂混合物的比率,混合之前储存在4℃下的鼠李糖脂JBR425的商品样品(来自Jeneil)与惰性吸附载体:硅藻土Celite 545(来自Celite Corp)。
经由使用配有1L提取器的Thar SFC-1000的超临界流体色谱法,以及在使用并行的两个提取器的全体积(16L)的Separex中试separex2X16L上,进行提取。
用被负载的鼠李糖脂混合物填充提取器。提取由两个步骤构成:在没有共溶剂的情况下使用超临界CO2的脱水/脱脂步骤,以及用超临界CO2和渐增比例的助溶剂除去单和二鼠李糖脂化合物的分级步骤。
脱脂步骤被延长至几乎没有油或水被提取。在第一步骤(超临界,但没有共溶剂)中,第一分离器主要含有游离脂肪酸且第二分离器含有水。在超临界加共溶剂步骤中,两个分离器的存在还增强了分级。
结果和分析
提取的收率
根据上述方法对级分进行质谱和ESI分析。图2和图3显示了每个级分的结果。

Claims (12)

1.一种从鼠李糖脂发酵混合物提取至少一种鼠李糖脂化合物的方法,所述方法包括以下步骤:
(a)将惰性吸附载体与鼠李糖脂发酵混合物混合;
(b)在高于31℃的温度和高于73巴的压力下将所述混合物溶解在有机溶剂中;
(c)分离至少一种脂肪化合物;
(d)向所述有机溶剂中加入共溶剂;
(e)改变温度和/或压力;
(f)分离第一鼠李糖脂化合物;和
(g)分离第二鼠李糖脂化合物。
2.根据权利要求1所述的方法,其中步骤(f)和(g)的鼠李糖脂提取是在共溶剂的加入(d)和步骤(e)中温度和/或压力的增加之后。
3.根据权利要求1或权利要求2所述的方法,其中脱脂步骤(c)在步骤(d)至(f)之前。
4.根据前述权利要求任一项所述的方法,其中所述步骤是按字母顺序排序的。
5.根据前述权利要求任一项所述的方法,其中在步骤(e)中所述温度和压力增加。
6.根据前述权利要求任一项所述的方法,其中所述第一鼠李糖脂化合物包含单鼠李糖脂。
7.根据前述权利要求任一项所述的方法,其中所述第二鼠李糖脂化合物包含二鼠李糖脂。
8.根据前述权利要求任一项所述的方法,其中所述惰性吸附载体包含硅藻土。
9.根据前述权利要求任一项所述的方法,其中所述有机溶剂是二氧化碳。
10.根据前述权利要求任一项所述的方法,其中所述有机溶剂处于超临界状态。
11.根据前述权利要求任一项所述的方法,其中所述有机溶剂在整个过程中处于超临界状态。
12.根据前述权利要求任一项所述的方法,其中所述共溶剂包含醇。
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