CN101163417A - 植物提取方法 - Google Patents
植物提取方法 Download PDFInfo
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- CN101163417A CN101163417A CNA2006800134512A CN200680013451A CN101163417A CN 101163417 A CN101163417 A CN 101163417A CN A2006800134512 A CNA2006800134512 A CN A2006800134512A CN 200680013451 A CN200680013451 A CN 200680013451A CN 101163417 A CN101163417 A CN 101163417A
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Abstract
本发明描述了用于产生植物提取物的方法，包括将植物材料与包含脂解酶的酶组合物一起温育。
Description
发明领域
本发明涉及用于提取植物化合物的改进的方法。
发明背景
许多植物化合物，例如多酚，具有期望的性质，例如作为抗氧化剂(antioxidant)、着色或调味剂(colouring or flavouring agent)。可将植物多酚添加至饮料(beverage)、乳制品(dairy product)、果酱(jam)、胶冻(jelly)、汤、果粥(fruit porridge)、蜜饯(preserve)和糖果产品。此外，认为植物多酚的高摄入降低冠心病的风险、降低胆固醇，还具有炎症作用(inflammatory effect)。因此用于提取这些多酚和其它植物化合物的改进的方法是高度期望的。
发明概述
目前本发明人发现，可通过将植物材料与脂解酶(lipolytic enzyme)，优选角质酶(cutinase)接触来促进植物化合物，例如来自植物材料的多酚的提取。
因此本发明在第一个方面提供产生植物提取物的方法，其包括将植物材料与包含脂解酶的酶组合物一起温育。
此外本发明在第二个方面提供一种方法，其包括将植物材料与包含脂解酶的酶组合物一起温育，和从所述植物材料分离水成液(aqueous liquid)。
此外本发明在第三个方面提供从植物材料提取化合物的方法，其包括将所述植物材料与包含脂解酶的酶组合物一起温育，和从所述植物材料分离水成液，所述液体包含所述化合物。
发明详述
通过用脂解酶，优选角质酶处理植物材料，能够将更多的植物固体溶解并且使其悬浮于水成液中。
从经酶处理的植物材料分离的水成液可以包含一种或多种化合物。所述化合物可以是存在于植物材料中的任何化合物。所述化合物可以是具有任何期望特性，例如抗氧化、着色或调味的期望化合物。所述化合物可以选自下组：5-(羟甲基)-2呋喃甲醛(5-(hydroxymethyl)-2furaldehyde)、苯甲酸(benzoeicacid)、肉桂酸；酚，优选苯甲酸衍生物或肉桂酸衍生物，更优选糖基苯甲酸(glycosyl benzoate)、糖基肉桂酸(glycosyl cinnamates)、水杨酸(salicylic acid)、异香草酸(isovanillic acid)、五倍子酸(gallic acid)、香豆酸(coumaric acid)、阿魏酸(ferulic acid)、咖啡酸(caffeic acid)、芥子酸(sinapic acid)、芥子碱(sinapicacid)；多酚，优选均二苯代乙烯(stilbene)衍生物或黄酮类(flavonoides)，更优选stilbeneol、黄酮醇(flavonol)、花色苷(anthocyanin)、异黄酮(isoflavone)、黄酮(flavon)，最优选stilbenol glycoside、黄烷醇糖苷(flavanol glycoside)、花色苷糖苷(anthocyanin glycoside)、异黄酮糖苷(isoflavon glycoside)、黄酮糖苷(flavon glycoside)、芪类(stilbene)、根皮苷(phloridzin)、白藜芦醇(resveratrol)、山奈酚(kaempferol)、槲皮素(quercetin)、异鼠李素(isorhamnetin)、杨梅黄素(myrecetin)、pelagonidin、花青素(cyanidin)、甲基花青素(peonidin)、翠雀定(delphinidin)、芹菜苷配基(apigenin)或木犀草素(luteolin)；萜类(terpenes)，优选类胡萝卜素(carotenoides)，更优选八氢番茄红素(phytoene)、链孢红素(neurosporene)、番茄红素(lycopene)、玉米胡萝卜素(zeacarotene)、胡萝卜素(carotene)；吡咯(pyrrols)，优选四吡咯(tetrapyrrol)，更优选四吡咯糖苷、叶绿素a/b(chlorophyll a/b)、叶绿素酸酯a/b(chlorophyllide a/b)、褐藻素a/b(pheophytin a/b)、脱镁叶绿酸a/b(pheophorbide a/b)或焦褐藻素a(pyropheophytin a)。
植物材料可以是任何植物材料，即植物性材料(vegetable material)，其包含期望的化合物。优选的植物材料可以源自浆果(berry)、梨果(pome)、柑橘类水果(citrus fruit)、核果(drupe)、蔬菜(vegetable)和种子(seed)。所述植物材料优选包含来自一种或多种选自下列的植物的材料：葡萄(红葡萄和绿葡萄)、酸果蔓的果实(cranberry)、黑醋栗(black currant)、红醋栗(red currant)、越橘(lingonberry)、接骨木(elderberry)、蓝莓(blueberry)、越桔(bilberry)、醋栗(gooseberry)、岩高兰(crowberry)、悬钩子(raspberry)、草莓(strawberry)、黑莓(blackberry)、猕猴桃(kiwi)、柠檬(lemon)、橙(orange)、莱檬(lime)、樱桃(cherry)、李子(plum)、桃(peach)、芒果(mango)、苹果(apple)、梨(pear)、胡萝卜(carrot)、黑胡萝卜(black carrot)、莴苣(lettuce)、甘蓝(cabbage)、红球甘蓝(red cabbage)、花椰菜(cauliflower)、椰菜(broccoli)、韭(leek)、芹菜(celery)、洋葱(onion)、大蒜(garlic)、人参(ginseng)、胡椒果实(pepper fruit)、红辣椒(chilli)、番茄(tomato)、茶(tea)、菠菜(spinach)、银杏(ginko biloba)、胡椒(pepper)(黑胡椒或白胡椒)、咖啡果(coffee berry)、咖啡豆(coffee bean)、油菜籽(rape seed)、芸苔(canola)。
当提取的期望化合物属于类胡萝卜素(例如，八氢番茄红素、链孢红素、番茄红素、玉米胡萝卜素或胡萝卜素)时，该化合物是pH稳定的，在提取步骤a)期间的pH不是关键的。然而，多数其它的期望化合物是pH不稳定的。pH不稳定化合物的实例是肉桂酰衍生物(cinnamoyl derivatives)、黄酮类(flavonoids)和黄酮苷(flavonoid glycoside)。因此优选在步骤a)的温育期间pH为优选不高于pH 7.0，不高于pH 6.5，或甚至不高于pH 6.0，如不高于pH 5.5。优选pH是至少3.0，至少3.5，或甚至至少4.0。
优选将植物材料在步骤a)之前或步骤a)期间浸软，以降低颗粒大小和增加提取。优选的还有来自果实加工的果肉(pulp)或果渣(pomace)，例如葡萄渣，如酿酒的副产品，其包含葡萄的种子和皮，并且化合物可以是多酚，如黄酮类。植物材料可以是来自果汁加工的残余物，即果肉，例如黑醋栗果肉或苹果果肉。同样优选作为植物材料的是植物的任何部分，包含蔬菜或水果的皮或种子，因为这些部分特别富含例如多酚和/或类胡萝卜素。
在步骤a)期间的温育之前或步骤a)期间的温育的同时，可将植物材料与第二酶，例如果胶酶，和/或纤维素酶接触。
可将本发明的方法整合至产生植物产品的方法中，所述植物产品如果汁(fruit juice)、果酱(jam)、胶冻(jelly)、汤(soup)、果粥(fruit porridge)、蜜饯(preserves)，由此增加植物产品中所期望化合物的含量。因此根据本发明，可将植物材料与额外的酶，例如果胶酶以及与脂解酶，优选角质酶一起温育。可将本发明的方法整合至酿酒方法中，由此增加葡萄汁中和发酵之后的酒(wine)中多酚的含量。
在实施方案中，将全部或基本上全部植物材料溶解并且使其悬浮于水成液中。
提取之后，可将期望化合物添加至或使用于饮料、乳制品、婴儿食品、汁(juice)、果酱、胶冻、汤、果粥、蜜饯和/或糖果点心产品(confectionary product)中。
酶
脂解酶
脂解酶是能够水解羧基酯键以释放羧酸或羧酸酯的酶(EC 3.1.1)，例如脂肪酶、磷脂酶、角质酶。
脂解酶可以是原核的，尤其是细菌的，或真核的，例如来自真菌或动物来源。脂解酶可以源自，例如以下属或种：嗜热霉属(Thermomyces)、细毛嗜热霉(T.lanuginosus)(也称作细毛腐质霉(Humicola lanuginosa))，腐质霉属(Humicola)、特异腐质霉(H.insolens)，镰孢属(Fusarium)、尖镰孢(F.oxysporum)、腐皮镰孢(F.solani)、异孢镰孢(F.heterosporum)，曲霉属(Aspergillus)、塔宾曲霉(A.tubigensis)、黑曲霉(A.niger)、米曲霉(A.oryzae)，根毛霉属(Rhizomucor)，念珠菌属(Candida)、C.antarctica、皱落念珠菌(C.rugosa)，青霉属(Penicillium)、沙门柏干酪青霉(P.camembertii)，根霉属(Rhizopus)、米根霉(Rhizopus oryzae)，犁头酶属(Absidia)、线筒菌属(Dictyostelium)，毛霉属(Mucor)，脉孢菌属(Neurospora)，根霉属、R.arrhizus、日本根霉(R.japonicus)，核盘菌属(Sclerotinia)，毛藓菌属(Trichophyton)，Whetzelinia，芽孢杆菌属(Bacillus)，柠檬酸杆菌属(Citrobacter)，肠杆菌属(Enterobacter)，爱德华氏菌属(Edwardsiella)，欧文氏菌属(Erwinia)，埃希氏菌属(Escherichia)、大肠杆菌(E.coli)，克雷伯氏菌属(Klebsiella)，变形菌属(Proteus)，普罗成登斯菌属(Providencia)，沙门氏菌属(Salmonella)，沙雷氏菌属(Serratia)，志贺氏菌属(Shigella)，链霉菌属(Streptomyces)，耶尔森氏菌属(Yersinia)，假单胞菌属(Pseudomonas)、洋葱假单胞菌(P.cepacia)，轮枝孢属(Verticillium)，壳针孢属(Septoria)和胶霉属(Gliocladium)。
脂解酶的一些具体实例如下所列：
来自蜜蜂(bee)或蛇毒(venom)或来自哺乳动物胰腺例如猪胰的磷脂酶。
来自米曲霉的磷脂酶(EP 575133、JP-A 10-155493)，来自Hyphozyma的磷脂酶(美国专利号6127137)。
来自细毛嗜热霉(也作细毛腐质霉)的脂肪酶(EP 305216、美国专利号5869438)、来自塔宾曲霉的脂肪酶(WO 9845453)、来自腐皮镰孢的脂肪酶(美国专利号5990069)。
来自尖镰孢的脂肪酶/磷脂酶(WO 98/26057)。
来自大刀镰孢的脂解酶(美国专利号5830736)或如WO 02/00852(PCT/DK 01/00448)或DK PA 200100304所述的脂解酶。
源自以上酶之一的通过取代、缺失或***一个或多个氨基酸的变体，例如WO 2000/32758，尤其是实施例54、5、6和13中所述，如来自细毛嗜热霉(也作细毛腐质霉)的脂肪酶的变体。
本发明优选的是在酸性pH具有活性的脂解酶，所述酸性pH例如在pH 7及以下，如pH 2至pH 7，例如pH 3至pH 5。
角质酶
优选脂解酶是角质酶。对于本发明而言，角质酶是归类于EC 3.1.1.74的酶。虽然用于本发明的角质酶可以是任何来源，包括哺乳动物、植物或动物来源，但优选所述角质酶是微生物来源的。具体而言所述角质酶是能够源自丝状真菌或酵母的角质酶。对于本发明优选的是在酸性pH具有活性的角质酶，所述酸性pH例如在pH 7及以下，如pH 2至pH 7，例如pH 3至pH 5。
优选角质酶源自特异腐质霉，例如由如下序列编码的角质酶或其片段：示于SEQ ID NO：1或SEQ ID NO：2的DNA序列，或与SEQ ID NO：1或SEQID NO：2中所示序列具有至少50％，优选至少60％，更优选至少70％，最优选至少80％，或甚至至少90％，如至少95％同一性的任何序列，所述片段具有角质酶活性。特别优选的是WO9613580的表A中所示的变体。
同样优选的是源自Fusarium solani f.sp.pisi(Nectria haematococca)的角质酶，例如根据WO 94/14964的角质酶。
多肽同一性
将术语“多肽同一性”理解为两种序列之间的同一性程度，以说明第一序列与第二序列的差别。同一性的程度可以依靠本领域已知的计算机程序，如在GCG程序包中提供的GAP来合适地测定(Program Manual for the WisconsinPackage，Version 8，August 1994，Genetics Computer Group，575 Science Drive，Madison，Wisconsin，USA 53711)(Needleman，S.B.and Wunsch，C.D.，(1970)，Journal of Molecular Biology，48，443-453。对于氨基酸序列比较使用以下设置：GAP产生罚分3.0和GAP延伸罚分0.1。用于同源性测定的氨基酸相关部分是成熟多肽，即无信号肽。
材料和方法
脂解酶和角质酶活性
脂解酶和/或角质酶活性可以使用三丁酸甘油酯为底物作为脂解活性测定。所述方法是基于酶水解三丁酸甘油酯以形成丁酸的速率。将丁酸用氢氧化物滴定并且将后者的消耗记录为时间的函数。
将一个脂肪酶单位(Lipase Unit；LU)定义为在标准条件下(即在30.0℃；pH 7.0；以Gum Arabic作为乳化剂和三丁酸甘油酯作为底物)每分钟释放1微摩尔可滴定丁酸的酶量。
更加详细描述这种分析方法的文件AF 95/5可向Novo Nordisk A/S，Denmark索取获得，在本文中将该文件作为参考并入。
酶
源自特异腐质霉的包含示于SEQ ID NO：2的多肽的角质酶组合物。
源自壳针孢属菌种(Septoria sp.)的角质酶组合物。
源自轮枝孢属菌种(Verticillium sp.)的角质酶组合物。
源自胶霉属菌种(Gliocladium.sp.)的角质酶组合物。
果胶酶，—包含多聚半乳糖醛酸酶活性26.000PG/ml的棘孢曲霉(Aspergillus aculeatus)菌株产生的组合物(Pectinex Ultra SP-L)。
实施例1
使用来自特异腐质霉的角质酶从黑醋栗果渣中提取多酚和花色素苷
将5.0克黑醋栗果渣与25.0mL 100mM乙酸缓冲液(acetate buffer)pH 5.8在50mL离心小管中混合，并加入1.00mL酶溶液(15000LU/ml)或空白，然后在40℃水浴中温育120分钟。所用酶是由本文中如SEQ ID NO：1所示的DNA序列编码的角质酶。温育之后，将样品在4℃于3500rpm离心15分钟，并且倾析出上清液。直接测量含糖量(°Brix)、浊度(NTU)和pH。将1mL上清液用0.1M乙酸稀释25倍并且在4℃放置过夜，然后在430和520nm用UV-Visual分光光度法评估颜色和花色素苷含量。Brown指数是520和430nm值之间的比率，并且显示花色素苷和总酚(total phenols)之间的比率。将全部样品进行两次重复操作。结果示于表1。将颜色结果相对于空白处理给出。
表1：黑醋栗果渣的提取
角质酶处理与空白相比，将花色素苷(O.D.520nm)的提取产率增加了12％，并且将全部多酚(O.D.430nm)的产率增加了17％。
实施例2
使用来自特异腐质霉的角质酶从苹果皮提取多酚和花色素苷
在50mL离心小管中称量5克苹果皮(Danish Ingrid Marie苹果)。添加含有所选酶的40mL 0.1M乙酸缓冲液pH 5.5，接着在40℃水浴中温育。120分钟之后，使用1.2μm真空滤器将样品过滤。用1M盐酸将pH调节至1.0并且将体积调节至50mL。在5℃储存过夜之后，通过分光光度法测量提取产率，使用在360nm的吸光度作为对总多酚产率的估计，并且使用在520nm的吸光度作为对花色素苷产率的估计。
使用以下酶组合(mL/kg苹果皮：空白不含酶；果胶酶(6.5PG(3.5)/g)；角质酶(6.5LU/g)；果胶酶(6.5PG(3.5)/g)+角质酶(6.5LU/g))。结果示于表2。将结果相对于空白处理给出。
表2：黑醋栗果渣的提取，相对于空白的产率
相对于空白样品，角质酶将多酚的提取产率增加了8％，并且将花色素苷的提取产率增加了28％。通过添加果胶酶和角质酶，将多酚产率相对于空白处理增加了19％。
实施例3
使用来自壳针孢属菌种的角质酶从苹果皮提取多酚和花色素苷
将10克苹果皮(Danish Ingrid Marie苹果)悬浮在含有所选酶的80mL 0.1M乙酸缓冲液pH 5.5中，接着在40℃水浴中温育10分钟，随后通过1.2微米(microM)滤器真空过滤。用1M盐酸将pH调节至1.0。在5℃储存过夜之后，通过分光光度法测量提取产率，使用在360nm的吸光度作为对总多酚产率的估计，并且使用在520nm的吸光度作为对花色素苷产率的估计。
使用以下酶组合：空白-不含酶；果胶酶(250mL/MT)；壳针孢属角质酶(8000LU/kg苹果皮)；果胶酶(0.25mL/kg苹果皮)+壳针孢属角质酶(8000LU/kg苹果皮)。结果示于表3。将结果相对于空白处理给出。
表3：苹果皮的提取，相对于空白的产率
单独的果胶酶不改变提取物中酚类(phenolics)的浓度。相对于空白样品，角质酶将多酚的提取产率增加了8％，并且将花色素苷的提取产率增加了21％。使用角质酶与果胶酶的组合将多酚产率进一步增加。
实施例4
分别使用来自轮枝孢属菌种和胶霉属菌种的角质酶从苹果皮提取多酚和花色素苷
如之前的实施例提取苹果皮(Danish Ingrid Marie苹果)，只是将2.5克苹果皮悬浮在20mL 0.1M乙酸缓冲液pH 5.5中。
使用以下酶组合：空白-不含酶；果胶酶(250mL/MT)；轮枝孢属角质酶(3200LU/kg苹果皮)；果胶酶(0.25mL/kg苹果皮)+轮枝孢属角质酶(3200LU/kg苹果皮)；胶霉属角质酶(1600LU/kg苹果皮)；果胶酶(0.25mL/kg苹果皮)+胶霉属角质酶(1600LU/kg苹果皮)。结果示于表4。将结果相对于空白处理给出。
表4：苹果皮的提取，相对于空白的产率
单独的果胶酶不增加酚类的提取。两种角质酶均将多酚的提取产率增加了＞20％，但不影响花色素苷的产率。将角质酶与果胶酶组合应用增加了多酚和花色素苷产率。
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Claims (16)
1.一种用于产生植物提取物的方法，其包括将植物材料与包含脂解酶的酶组合物一起温育。
2.一种方法，其包括
a.将植物材料与包含脂解酶的酶组合物一起温育，和
b.从所述植物材料分离水成液。
3.一种用于从植物材料提取化合物的方法，其包括
a.将所述植物材料与包含脂解酶的酶组合物一起温育，所述脂解酶优选角质酶，和
b.从所述植物材料分离水成液，所述液体包含所述期望化合物。
4.权利要求1至3任一项的方法，其中在步骤a)的温育期间pH为pH3.0至7.0。
5.权利要求1至4任一项的方法，其中所述化合物是着色化合物、抗氧化剂、芳香化合物和/或脂质。
6.权利要求1至5任一项的方法，其中所述化合物选自下组：三-、二-和单酸甘油酯、脂肪酸、花色素苷、丹宁、前花色素、均二苯代乙烯类、白藜芦醇、桂皮酸衍生物、苯甲酸衍生物、叶绿素、黄酮类、五倍子酸、黄烷-3醇(flavan-3ols)，黄酮醇、根皮苷、肉桂酸和羟甲基糠醛。
7.权利要求1至6任一项的方法，其中所述植物材料是浆果：例如葡萄(红葡萄和绿葡萄)、酸果蔓的果实、黑醋栗、越橘(lingonberry)、红醋栗、接骨木、蓝莓、越桔(bilberry)、鹅莓、岩高兰、悬钩子、草莓、黑莓、猕猴桃，柑橘类果实：例如柠檬、橙、莱檬，核果：例如樱桃、李子、桃、芒果，梨果：例如苹果、梨，蔬菜：例如胡萝卜、黑胡萝卜、莴苣、甘蓝、红球甘蓝、花椰菜、椰菜、韭、芹菜、洋葱、大蒜、人参、胡椒果实、红辣椒、番茄、茶、菠菜、银杏(ginko biloba)，种子：如胡椒(例如黑胡椒或白胡椒)、咖啡果、咖啡豆、油菜籽、芸苔。
8.权利要求1至7任一项的方法，其中所述脂解酶是角质酶。
9.权利要求1至8任一项的方法，其中所述角质酶源自腐质霉属，轮枝孢属或胶霉属之内的菌种。
10.权利要求1至9任一项的方法，其中所述角质酶源自特异腐质霉。
11.权利要求1至10任一项的方法，其中所述角质酶包含与SEQ ID NO：1或SEQ ID NO：2所示序列或其具有角质酶活性的片段具有至少50％同一性的氨基酸序列。
12.权利要求1至11任一项的方法，其包括在步骤a)之前或步骤a)期间将植物材料浸软。
13.权利要求1至12任一项的方法，其中所述植物材料是或包含果渣和/或来自果实加工的废材料。
14.权利要求1至13任一项的方法，其中所述植物材料包含水果或蔬菜的皮。
15.权利要求1至14任一项的方法，其中在步骤a)的温育之前或与步骤a)的温育同时将植物材料与果胶酶接触。
16.权利要求1至15任一项的方法，其中所述植物材料包含葡萄，并且将所述水成液用酵母发酵以产生酒。
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| JPH01300848A (ja) * | 1988-05-27 | 1989-12-05 | Osaka Gas Co Ltd | 茶葉の処理方法 |
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