CN101296626B - 基于肉的食品 - Google Patents
基于肉的食品 Download PDFInfo
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- CN101296626B CN101296626B CN2006800400900A CN200680040090A CN101296626B CN 101296626 B CN101296626 B CN 101296626B CN 2006800400900 A CN2006800400900 A CN 2006800400900A CN 200680040090 A CN200680040090 A CN 200680040090A CN 101296626 B CN101296626 B CN 101296626B
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- Prior art keywords
- meat
- phosphatidase
- phospholipase
- food
- enzyme
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Abstract
本发明涉及通过用磷脂酶处理肉来产生基于肉的食品的方法、能够通过本发明的方法获得的基于肉的食品和磷脂酶用于制备基于肉的食品的用途。
Description
技术领域
本发明涉及通过用磷脂酶处理肉来生产的基于肉的食品(meat based foodproduct),和用于生产该食品的方法。
发明背景
肉含有脂肪,并且期望在基于肉的肉制品中脂肪是稳定的,从而将脂肪损失(例如烹饪过程中的脂肪损失)保持在低水平,并且将可见的游离脂肪的量减少。在乳化肉制品(emulsified meat product)中,可以添加乳化剂以达到这些效果。此外,期望肉汁(meat juice)损失低,并且有期望的风味(taste)、质地(texture)和外观(appearance)。
众所周知能够以不同的方式使肉制品中的脂肪稳定。一种方法是添加分离的蛋白质或蛋白质浓缩物,例如,酪蛋白酸钠(Na-caseinate)、乳清蛋白浓缩物或大豆蛋白分离物或浓缩物。已经开发了特殊的技术以确保充分乳化,如在添加和斩拌(chop)其它成分之前,用肉配方中的脂肪组分乳化蛋白。所述蛋白的特征在于相对昂贵,并且在肉制品中的允许量有限。添加剂如甘油一酸酯和甘油二酸酯(mono and di-glycerides)和它们的柠檬酸酯是通常用于肉加工中的另一种技术。它们是有效的乳化剂,但是由于价格或不希望肉制品标签上存在添加剂,它们的应用通常是不理想的。
Dacaranhe和Terao(Dacaranhe,CD and Terao,J:Effect of cabbagephospholipase D treatment on the oxidative stability of beef homogenate and eggyolk phosphatidylcholine liposomes.Journal of Food Science 67(2002)2619-2624)和Chung-Wang等(Chung-Wang,YJ et al.:Reduced oxidation of freshpork in the presence of exogenous hydrolases and bacteria at 2℃.Journal ofApplied Microbiology 82(1997)317-324)已经显示磷脂酶处理增加原料肉的氧化稳定性。
发明概述
发明者发现由经磷脂酶处理的肉制成的基于肉的食品,与由未经磷脂酶处理的相似的肉制成的相似的基于肉的食品相比可具有改进的性质。因此,本发明涉及用于生产基于肉的食品的方法,其包括:a)将肉与磷脂酶接触;b)加热经磷脂酶处理的肉;和c)由所述经磷脂酶处理的肉生产食品;其中步骤b)在步骤c)之前、期间或之后进行。在另外的方面,本发明涉及磷脂酶用于生产基于肉的食品的用途,和能够通过本发明的方法获得的基于肉的食品。
发明详述
根据本发明的基于肉的食品是基于肉并且适于用作人或动物的食物的任何产品。在本发明的一个实施方式中,基于肉的食品是用于喂养动物的饲料产品,例如宠物食品。在另一个实施方式中,基于肉的食品是小吃产品(snackproduct)。
肉
根据本发明的肉是源自任何种类动物的任何种类的组织。根据本发明的肉可以是包含源自动物的肌肉纤维的组织。在本发明的一个实施方式中,肉是动物肌肉,例如,完整的动物肌肉或从动物肌肉切下的肉块。在另一个实施方式中,根据本发明的肉包含动物的内部器官,例如心、肝、肾、脾、胸腺和脑。可以通过本领域已知的任何其它适当的方法将肉磨碎(grind)或绞碎(mince)或切割(cut)成较小的块。根据本发明的肉可源自任何种类的动物,例如,源自牛、猪、羔羊、绵羊、山羊、鸡、母鸡、火鸡、鸵鸟、雉(pheasant)、鹿、麋(elk)、驯鹿(reindeer)、水牛、野牛(bison)、羚羊、骆驼、袋鼠;任何种类的鱼,例如黍鲱(sprat)、鳕(cod)、黑线鳕(haddock)、鲔鱼(tuna)、海鳗(sea eel)、鲑鱼(salmon)、鲱鱼(herring)、沙丁鱼(sardine)、鲭(mackerel)、竹荚鱼(horsemackerel)、秋刀鱼(saury)、圆腹鲱(round herring)、青鳕(Pollack)、鲽鱼(flatfish)、鳀鱼(anchovy)、沙丁鱼(pilchard)、小鳍鳕(blue whiting)、pacific whithing、鳟鱼(trout)、鲇鱼(catfish)、鲈鱼(bass)、毛鳞鱼(capelin)、枪鱼(marlin)、紫红笛鲷(red snapper)、挪威鳕(Norway pout)和/或狗鳕(hake);任何种类的甲壳类,例如蛤(clam)、贻贝(mussel)、扇贝(scallop)、鸟蛤(cockle)、玉黍螺(periwinkle)、蜗牛(snail)、牡蛎(oyster)、小虾(shrimp)、龙虾(lobster)、小龙虾(langoustine)、蟹(crab)、淡水小龙虾(crayfsh)、墨鱼(cuttlefish)、乌贼(squid)和/或章鱼(octopus)。在本发明的一个实施方式中,肉是牛肉、猪肉、鸡肉和/或火鸡肉。在另一个实施方式中,肉是鱼肉。
根据本发明的基于肉的食品是基于肉的任何食品。基于肉的食品可包含非肉成分例如水、盐、面粉、乳蛋白、植物蛋白、淀粉、水解蛋白、磷酸盐/酯(phosphate)、酸和/或香料(spices)。根据本发明的基于肉的食品可包含至少30%(重量/重量)肉,例如至少50%,至少60%或至少70%肉。
在一个实施方式中,基于肉的食品是加工的肉制品,例如香肠(sausage)、波洛尼亚香肠(bologna)、肉糕(meat loaf)、碎肉制品(comminuted meat product)、绞肉(ground meat)、培根(bacon)、半熟干香肠(polony)、萨拉米香肠(salami)或肉饼(pate)。加工的肉制品可进一步包含例如盐、香料、乳蛋白、植物性成分、着色剂和/或增稠剂(texturising agent)。加工的肉制品可以是乳化的肉制品,例如由基于肉的乳化物(emulsion)制造的乳化的肉制品,例如摩泰台拉香肚(mortadella)、波洛尼亚香肠、加香料的意大利香肠(pepperoni)、肝肠(liversausage)、鸡肉香肠(chicken sausage)、维也纳香肠(wiener)、法兰克福香肠(frankfurter)、午餐肉(luncheon meat)、肉饼(meat pate)。可以将基于肉的乳化物烹制、灭菌或焙烤,例如以焙烤形式或在填入包衣(casing)之后再进行烹制、灭菌或焙烤,所述包衣例如塑料、胶原、纤维素或天然包衣。加工肉制品还可以是重构肉制品(reconstructed meat product),例如重构火腿。本发明的肉制品可以经过加工步骤例如盐腌,例如干盐腌;腌制(curing),例如盐水腌渍(brinecuring);干燥;熏制;发酵;烹制;罐藏;甑馏(retorting);切片(slicing);和/或切碎(shredding)。
生产基于肉的食品的方法
可以通过将肉与磷脂酶接触,并且由经处理的肉生产基于肉的食品来产生根据本发明的基于肉的食品。在与磷脂酶接触时,肉将通常是生肉,但也可以是在与磷脂酶接触之前经过例如热处理、预烹制或照射的肉。也可将肉在与磷脂酶接触之前冷冻。可以通过向肉添加磷脂酶,例如纯化的磷脂酶来实现肉与磷脂酶接触。可以通过将肉,例如肉块、绞碎的肉或基于肉的乳化物与磷脂酶和可应用的用于形成基于肉的食品的其它成分以本领域已知的任何方法混合来实现肉与磷脂酶的接触。在与肉接触之前,可将磷脂酶与其它成分例如水、盐、面粉、乳蛋白、植物蛋白、淀粉、水解蛋白、磷酸盐/酯、酸和/或香料混合,例如以形成腌泡汁(marinade)或腌渍液(pickling liquid)。可以调节腌泡汁中磷脂酶的量从而在基于肉的食品中达到期望的磷脂酶的最终量。可通过用包含磷脂酶的腌泡汁腌泡和/或用转筒混合(tumbling)来实现肉例如完整的动物肌肉或动物肌肉块与磷脂酶的接触。如果肉制品是加工的肉制品,例如乳化的肉制品,可以将磷脂酶例如混入基于肉的乳化物,或混入用于形成加工的肉制品的任何其它形式的基于肉的混合物。在本发明的一个实施方式中,将磷脂酶添加至腌泡汁或腌渍液。可以通过用本领域已知的用于注射和/或泵送液体至肉中的任何方法将包含磷脂酶的液体注射和/或泵送到肉中来将磷脂酶添加至肉。本发明的基于肉的食品可以是已经与磷脂酶接触并且包装的(packed)肉块。
在本发明的方法中,将肉在与磷脂酶接触后进行热处理。可以通过本领域已知的任何方法进行加热。加热将通常作为基于肉的食品的生产中的整合部分来进行,例如通过烹制进行,但是也可在形成基于肉的食品所需要的加工步骤之前进行,或在基于肉的食品形成之后进行。因此加热可以在产生基于肉的食品之前、期间或之后进行。可以将肉与磷脂酶接触并且使磷脂酶水解磷脂之后再烹制根据本发明的基于肉的食品。烹制可以通过本领域已知的任何方法进行,例如在熏制室(smoking cabinet)中,在沸水中,在水浴中,在烤箱中,通过微波加热,通过烤炙(grilling),通过加压蒸煮,通过甑馏和/或通过煎炸。在本发明的一个实施方式中,将基于肉的食品加热至例如温度为50-140℃,例如60-120℃、60-100℃或70-100℃。在另一个实施方式中,将基于肉的食品加热至足以使磷脂酶失活的温度并且保持足以使磷脂酶失活的时间。在更进一步的实施方式中,将磷脂酶处理的肉加热到至少50℃,例如至少60℃,或至少70℃。
本发明的基于肉的食品可以用于直接消费,或可在消费之前使其经过进一步的加工。可将本发明的基于肉的食品用作产生其它食品的原料或成分。
本发明的方法中使用的酶
磷脂例如卵磷脂(lecithin)或磷脂酰胆碱(phosphatidylcholine),由在外部(sn-1)和中部(sn-2)位置用两个脂肪酸酯化并且在第三位置用磷酸酯化的甘油组成;而所述磷酸又可酯化为氨基醇。磷脂酶是参与磷脂水解的酶。能够区分几种类型的磷脂酶活性,包括磷脂酶A1和A2(通称为磷脂酶A),其水解一个脂肪酰基(分别在sn-1和sn-2位置)以形成溶血磷脂。磷脂酶B则水解溶血磷脂中剩余的脂肪酰基。
本发明的方法中使用的酶包括磷脂酶,例如磷脂酶A1、磷脂酶A2、磷脂酶B、磷脂酶C或磷脂酶D。在本发明的方法中,磷脂酶处理可由一种或多种磷脂酶提供,例如两种或更多种磷脂酶,例如两种磷脂酶,包括但不限于:用A和B两种类型处理;用A1和A2两种类型处理;用A1和B两种类型处理;用A2和B两种类型处理;或用相同类型的两种或更多种不同的磷脂酶处理。还包括用一种类型的磷脂酶处理,例如A1、A2、B、C或D。
根据标准酶EC分类将磷脂酶A1定义为EC 3.1.1.32。
官方名称:磷脂酶A1。
催化的反应:
磷脂酰胆碱+水<=>2-酰基甘油磷酸胆碱+脂肪酸阴离子
注释:与EC 3.1.1.4相比具有广泛得多的特异性。
根据标准酶EC分类将磷脂酶A2定义为EC3.1.1.4
官方名称:磷脂酶A2。
别名:磷脂酰胆碱2-酰基水解酶。
卵磷脂酶a;磷脂酶(phosphatidase);或磷脂酶(phosphatidolipase)。
催化的反应:
磷脂酰胆碱+水<=>1-酰基甘油磷酸胆碱+脂肪酸阴离子
注释:还作用于磷脂酰乙醇胺、胆碱缩醛磷脂(choline plasmalogen)和磷脂,去除连于(attach to)2位的脂肪酸。
根据标准酶EC分类将磷脂酶B定义为EC 3.1.1.5
官方名称:溶血磷脂酶。
别名:卵磷脂酶b;溶血卵磷脂酶;
磷脂酶B;或PLB。
催化的反应:
2-溶血磷脂酰胆碱+水<>甘油磷酸胆碱+脂肪酸阴离子
根据标准酶EC分类将磷脂酶C定义为EC 3.1.4.3。磷脂酶C水解磷脂酰胆碱和其它甘油磷脂(例如磷脂酰乙醇胺)上的磷酸键,生成二酰基甘油;该酶还会水解鞘磷脂(sphingomyelin)、心磷脂(cardiolipin)、胆碱缩醛磷脂和神经酰胺磷脂(ceramide phospholipids)的磷酸键。
与磷脂酰胆碱的反应:
磷脂酰胆碱+水<=>1,2-二酰基甘油+磷酸胆碱
根据标准酶EC分类将磷脂酶D定义为EC 3.1.4.4。磷脂酶D水解磷脂和鞘磷脂的磷酸键以产生相应的磷脂酸。
与磷脂酰胆碱的反应:
磷脂酰胆碱+水<=>胆碱+磷脂酸(phosphatidate)
磷脂酶A
磷脂酶A活性可以由也具有其它活性的酶提供,例如具有磷脂酶A活性的脂肪酶。例如磷脂酶A活性可以来自具有磷脂酶副活性(side activity)的脂肪酶。在本发明的另一个实施方式中,磷脂酶A酶活性由基本上仅具有磷脂酶A活性的酶提供,并且其中所述磷脂酶A酶活性不是副活性。
磷脂酶A可以是任何来源,例如动物(例如,哺乳动物)来源,例如来自胰(例如牛或猪胰),或蛇毒或蜂毒。或者,磷脂酶A可以是微生物来源,例如来自丝状真菌、酵母或细菌,例如以下菌属或菌种:曲霉属(genusAspergillus),例如黑曲霉(A.niger);网柄菌属(Dictyostelium),例如盘基网柄菌(D.discoideum);毛霉属(Mucor),例如爪哇毛霉(M.javanicus)、大毛霉(M.mucedo)、细孢毛霉(M.subtilissimus);脉孢菌属(Neurospora),例如粗糙脉孢菌(N.crassa);根毛菌属(Rhizomucor),例如微小根毛霉(R.pusillus);根霉属(Rhizopus),例如少根根霉(R.arrhizus)、日本根霉(R.japonicus)、匍枝根霉(R.stolonifer);核盘菌属(Sclerotinia),例如大豆核盘菌(S.libertiana);毛藓菌属(Trichophyton),例如红色毛藓菌(T.rubrum);Whetzelinia菌属,例如W.sclerotiorum;芽孢杆菌属(Bacillus),例如巨大芽孢杆菌(B.megaterium)、枯草芽孢杆菌(B.subtilis);柠檬酸杆菌属(Citrobacter),例如弗式柠檬酸杆菌(C.freundii);肠杆菌属(Enterobacter),例如产气肠杆菌(E.aerogenes)、阴沟肠杆菌(E.cloacae);爱德华氏菌属(Edwardsiella),迟钝爱德华氏菌(E.tarda);欧天氏菌属(Erwinia),例如草生欧文氏(E.herbicola);埃希氏菌属(Escherichia),例如大肠杆菌(E.coli);克雷伯氏菌属(Klebsiella),例如肺炎克雷伯氏菌(K.pneumoniae);变形菌属(Proteus),例如普通变形菌(P.vulgaris);普罗威登斯菌属(Providencia),例如斯氏普罗威登斯菌(P.stuartii);沙门氏菌(Salmonella),例如鼠伤寒沙门氏菌(S.typhimurium);沙雷氏菌属(Serratia),例如液化沙雷氏菌(S.liquefasciens)、粘质沙雷氏菌(S.marcescens);志贺氏菌属(Shigella),例如弗氏志贺氏菌(S.flexneri);链霉菌属(Streptomyces),例如S.violeceoruber,耶尔森氏菌属(Yersinia),例如小肠结肠炎耶尔森氏菌(Y.enterocolitica)。由此,磷脂酶A可以是真菌的,例如源自核菌(Pyrenomycetes)类,例如镰孢属(genusFusarium),例如大刀镰孢(F.culmorum)、异孢镰孢(F.heterosporum)、腐皮镰孢(F.solani)的菌株或尖镰孢(F.oxysporum)的菌株。磷脂酶A还可以来自曲霉属内的丝状真菌菌株,例如泡盛曲霉(Aspergillus awamori)、臭曲霉(Aspergillusfoetidus)、日本曲霉(Aspergillus japonicus)、黑曲霉或米曲霉(Aspergillu oryzae)的菌株。优选的磷脂酶A源自镰孢属的菌株,尤其是F.venenatum或尖镰孢,例如来自WO 98/26057中所述菌株DSM 2672,特别在WO 98/26057的权利要求36和SEQ ID NO.2中所述。另一个优选的磷脂酶A是来自链霉菌属的PLA2,例如来自S.violaceoruber的PLA2。在另外的实施方式中,磷脂酶是如WO 00/32758(Novozymes A/S,Denmark)中公开的磷脂酶。
可以将A型磷脂酶的活性例如以Lecitase单位(LEU)表示。使用卵磷脂作为底物相对于磷脂酶标准物来测量Lecitase单位的磷脂酶活性。磷脂酶A催化卵磷脂水解为溶血卵磷脂和游离脂肪酸。将释放的脂肪酸用0.1N氢氧化钠在标准条件(pH 8.00;40.00℃±0.5)下滴定。测定在脂肪酸的中和过程中氢氧化钠的消耗的速率作为磷脂酶A的活性,并且相对于Lecitase(磷脂酶)标准物(可从Novozymes A/S,Denmark获得)表示为Lecitase单位(LEU)。将1LEU定义为标准条件(pH 8.00;40.00℃±0.5)下与稀释至1LEU/g标称活性的Lecitase标准物产生相同的氢氧化钠消耗速率(μmol/min)的酶量。
磷脂酶B
术语“磷脂酶B”用于本文中与本发明的酶相关时意欲包括具有磷脂酶B活性的酶。
磷脂酶B活性可以由也具有其它活性的酶提供,例如具有磷脂酶B活性的脂肪酶。磷脂酶B活性可以例如来自具有磷脂酶B副活性的脂肪酶。在本发明的另一个实施方式中,磷脂酶B酶活性由基本上仅具有磷脂酶B活性的酶提供,并且其中所述磷脂酶B酶活性不是副活性。在本发明的一个实施方式中,磷脂酶B不是如WO 98/26057中限定的具有磷脂酶B副活性的脂肪酶。
磷脂酶B可以是任何来源,例如动物(例如哺乳动物)来源,例如来自肝(例如大鼠肝)。或者,磷脂酶B可以是微生物来源,例如来自丝状真菌、酵母或细菌,例如曲霉属或种,例如臭曲霉、烟曲霉(A.fumigatus)、构巢曲霉(A.nidulans)、黑曲霉、米曲霉;葡萄孢属(Botrytis),例如灰葡萄孢(B.cinerea);念珠菌属(Candida),例如白念珠菌(C.albicans);隐球菌属(Cryptococcus),例如新生隐球菌(C.neoformans),埃希氏菌属,例如大肠杆菌,镰孢属,例如拟分枝孢镰孢(F.sporotrichioides)、F.venenatum、轮状镰孢(F.verticillioides);Hyphozyma;克鲁维酵母属(Kluyveromyces),例如乳酸克鲁维酵母(K.lactis);Magnaporte,例如M.grisea;绿僵菌属(Metarhizium),例如绿僵菌(M.anisopliae);球腔菌属(Mycosphaerella),例如禾生球腔菌(M.graminicola);脉孢菌属,例如粗糙脉孢菌;青霉属(Penicillium),例如特异青霉(P.notatum);酵母属(Saccharomyces),例如酿酒酵母(S.cerevisiae);裂殖酵母属(Schizosaccharomyces),例如粟酒裂殖酵母(S.pombe);有孢圆酵母属(Torulaspora),例如戴尔有孢圆酵母(T.delbrueckii);弧菌属(Vibrio);例如霍乱弧菌(V.cholerae)。优选的磷脂酶B源自曲霉属的菌株,尤其是源自黑曲霉的磷脂酶LLPL-1或LLPL-2,例如大肠杆菌克隆DSM 13003或DSM 13004中所含的,或源自米曲霉的磷脂酶LLPL-1或LLPL-2,如WO 01/27251中描述的大肠杆菌克隆DSM 13082或DSM 13083中所含的,特别是WO 01/27251的权利要求1和SEQ ID NOs.2、4、6或8中所描述的。
磷脂酶C
磷脂酶C活性可以由也具有其它活性的酶提供,例如具有磷脂酶C活性的脂肪酶或具有磷脂酶C活性的磷酸酶。磷脂酶C活性可以例如来自具有磷脂酶C副活性的脂肪酶。在本发明的其它实施方式中,磷脂酶C酶活性由基本上仅具有磷脂酶C活性的酶提供,并且其中所述磷脂酶C活性不是副活性。
磷脂酶C可以是任何来源的,例如动物来源,例如哺乳动物来源的,植物来源的或微生物来源,例如真菌来源或细菌来源的,例如来自分枝杆菌属(Mycobacterium)菌株,例如结核分枝杆菌(M.tuberculosis)或牛结核分枝杆菌(M.bovis);芽孢杆菌属菌株,例如蜡样芽孢杆菌(B.cereus);梭菌属(Clostridium)菌株,例如双酶梭菌(C.bifermentans)、溶血梭菌(C.haemolyticum)、诺氏梭菌(C.novyi)、索氏梭菌(C.sordellii)或产气荚膜梭菌(C.perfringens);利斯特氏菌属(Listeria)菌株,例如单核细胞增生利斯特菌(L.monocytogenes);假单胞菌属(Pseudomonas)菌株,例如铜绿假单胞菌(P.aeruginosa);或葡萄球菌属(Staphylococcus)菌株,例如金黄色葡萄球菌(S.aureus);或伯克霍尔德氏菌属(Burkholderia)菌株,例如类鼻疽伯克霍尔德氏菌(B.pseudomallei)。
磷脂酶D
磷脂酶D活性可以由也具有其它活性的酶提供,例如具有磷脂酶D活性的脂肪酶、具有磷脂酶D活性的磷酸酶或具有磷脂酶D活性的胆碱酯酶。磷脂酶D活性可以例如来自具有磷脂酶D副活性的脂肪酶。在本发明的其它实施方式中,磷脂酶D酶活性由基本上仅具有磷脂酶D活性的酶提供,并且其中所述磷脂酶D酶活性不是副活性。
磷脂酶D可以是任何来源的,例如动物来源,例如哺乳动物来源的,例如来自小鼠、大鼠或中国仓鼠;植物来源的,例如来自甘蓝、玉米、稻、蓖麻(castor bean)、烟草、豇豆(cowpea)或拟南芥(Arabidopsis thaliana);或微生物来源的,例如细菌来源的,例如来自棒杆菌属(Corynebacterium)菌株,例如假结核棒杆菌(C.pseudotuberculosis)、溃疡棒杆菌(C.ulcerans)或溶血棒杆菌(C.haemolyticum);或真菌来源的,例如来自链霉菌属的菌株,例如抗生链霉菌(S.antibioticus)或色褐链霉菌(S.chromofuscus);木霉属(Trichoderma)的菌株,例如里氏木霉(T.reesei);酵母属的菌株,例如酿酒酵母;或曲霉属的菌株,例如米曲霉、黑曲霉、构巢曲霉或烟曲霉。
酶来源和剂型(formulation)
本发明的方法中使用的磷脂酶可以源自或可获得自本文提到的任何来源。术语“源自”在本上下文中的意思是酶可分离自天然存在该酶的生物,即所述酶的氨基酸序列的特征(identity)与天然酶一样。术语“源自”还指酶可在宿主生物中重组产生,所述重组产生的酶具有与天然酶一样的特征或具有修饰的氨基酸序列,例如缺失、插入和/或取代一个或多个氨基酸,即重组产生的酶是天然氨基酸序列的突变体和/或片段。天然酶的意思包括天然变体。此外,术语“源自”包括通过例如肽合成等合成产生的酶。术语“源自”还包括经修饰的酶,例如通过糖基化、磷酸化等,无论体内或体外修饰的酶。术语“可获得”在本上下文中的意思是酶具有与天然酶一致的氨基酸序列。所述术语包括已经从天然存在该酶的生物分离的酶,或在相同类型的生物或其它生物中重组表达的酶,或通过例如肽合成等合成产生的酶。关于重组产生的酶,术语“可获得”和“源自”指酶的同一性,而非重组产生该酶的宿主生物的同一性。
因此,可以通过使用任何合适的技术从微生物获得磷脂酶。例如,可以用本领域已知的方法通过发酵合适的微生物和随后从所得发酵液或微生物分离磷脂酶制备物来获得磷脂酶酶制备物。还可以通过使用重组DNA技术来获得磷脂酶。这种方法通常包括:培养用重组DNA载体转化的宿主细胞,所述重组DNA载体包含编码所述磷脂酶的DNA序列,并且所述DNA序列与适当的表达信号可操作地连接,由此能够在允许表达所述酶的条件下在培养基中表达磷脂酶;和从培养物回收酶。还可以将DNA序列并入宿主细胞的基因组。DNA序列可以是基因组、cDNA或合成来源的或这些来源任何组合,并且可根据本领域已知的方法分离或合成。
合适的磷脂酶是商业上可获得的。实际应用中酶的实例是例如(Novozymes A/S,Denmark)或(Novozymes A/S,Denmark and Chr.Hansen A/S,Denmark)。合适的磷脂酶B是例如黑曲霉磷脂酶LLPL-2,其能够在黑曲霉中重组产生,如WO 01/27251中所述。
在本发明的方法中可将磷脂酶纯化。术语“纯化”用于本文包括不含(freefrom)来自生物的成分的磷脂酶酶蛋白,所述磷脂酶酶蛋白源自所述生物。术语“纯化”还包括不含来自天然生物的成分的磷脂酶酶蛋白,所述磷脂酶酶蛋白从该天然生物获得,这也被称为“基本上纯的(essentially pure)”磷脂酶,并且可与天然存在且未经遗传修饰的磷脂酶特别相关,所述遗传修饰如通过缺失、取代或插入一个或多个氨基酸残基来进行。
因此,可将磷脂酶纯化,即只存在较少量其它蛋白质。表述“其它蛋白质”具体涉及其它酶。本文所用术语“纯化”还指去除其它成分,特别是其它蛋白质,并且最特别是存在于磷脂酶来源的细胞中的其它酶。磷脂酶可以是“基本上纯的”,即不含来自产生该磷脂酶的生物的其它成分,所述产生该磷脂酶的生物即,例如,用于重组产生磷脂酶的宿主生物。优选地,所述酶是至少75%(重量/重量)纯的,更优选至少80%、85%、90%或甚至至少95%纯的。在还更优选的实施方案中,所述磷脂酶是至少98%纯的酶蛋白制剂。
术语“磷脂酶”包含对于所述酶的催化活性是必需的任何辅助化合物(auxiliary compound),例如合适的受体或辅因子(cofactor),其可以是或可以不是天然存在于反应体系中的。
磷脂酶可以是适于所述用途的任何形式,例如以下形式:干粉末或颗粒(granulate)、无粉尘的颗粒、液体、稳定的液体(stabilized liquid)或保护的酶。可以例如US 4,106,991和US 4,661,452所公开的来产生颗粒,并且可以任选地通过本领域已知的方法来涂覆它们。例如,可以根据已确定的方法通过添加稳定剂如糖、糖醇或其它多元醇(polyol)、乳酸或其它有机酸来使液体酶制备物稳定。保护的酶可以根据EP 238,216中公开的方法制备。
通过本发明的方法,作为用磷脂酶处理肉的结果,可通过水解磷脂酰胆碱加磷脂酰乙醇胺总量的至少5%,例如至少10%、至少20%、至少30%或至少50%来修饰肉的磷脂。在一个实施方式中,作为磷脂酶处理的结果,可将肉中磷脂的至少5%,例如至少10%、至少20%、至少30%或至少50%水解。
通过本发明的方法,获得的基于肉的食品中的磷脂含量可以是将肉与磷脂酶接触之前肉中存在的磷脂总含量的少于90%,例如少于80%,例如少于60%或少于50%。可通过技术人员已知的任何方法(例如通过HPLC)来测量磷脂含量。
在本发明的方法中使用的磷脂酶量可以取决于具体处理条件下具体磷脂酶对肉中存在的磷脂的活性。磷脂酶的量可以由技术人员通过本领域用于优化酶反应的已知方法来测定,例如通过测定达到期望的肉磷脂水解程度和/或达到对基于肉的食品的特性的期望效果所需的磷脂酶量。
当使用磷脂酶A时,磷脂酶的量可以是例如0.1-50LEU每克脂肪,例如0.5-25或1-10LEU每克脂肪。
通过本发明的方法产生的基于肉的食品与无肉的磷脂酶处理而生产的类似的基于肉的食品相比可具有改进的性质,例如根据本发明的肉制品可具有改进的脂肪稳定性、更少量的可见脂肪、增加的水结合和/或改进的质地性质(textural properties),例如增加的硬度(firmness)。
在一个实施方式中本发明涉及磷脂酶用于生产基于肉的食品的用途,并且在进一步的实施方式中本发明涉及通过本发明的方法获得的或可获得的基于肉的食品。
实施例
实施例1
从以下成分(%重量/重量)产生200g肉制品:
60%绞碎的牛肉,含18%脂肪
1.8%NaCl
0.4%三聚磷酸钠
37.8%水
在食品加工机中将温度为5℃的牛肉和NaCl混合1分钟,添加温度为5℃的水和磷酸盐并且继续混合3分钟。将100g转移至用于烹制的容器。如下进行烹制:将容器中的混合物加热至40℃并将该温度保持30分钟,其后将容器中的混合物置于90℃水浴并且保持直至中心温度达到75℃。将产物冷却至5℃并且在次日评估。
制备一个不添加磷脂酶的对照样品。
对于样品1和样品2,磷脂酶均与NaCl一起添加至肉。
结果示于表1。
表1
脂肪外观 | 肉汁外观 | |
对照样品 | 大脂肪颗粒 | 浅褐色 |
样品1 | 与对照样品相比大脂肪颗粒少而小脂肪颗粒多 | 褐色 |
样品2 | 与对照样品相比小的脂肪颗粒 | 中度褐色 |
实施例2
用如表2中描述的组成以实验室规模制造午餐肉。将猪肉和脂肪切块(dize)(大约3×3cm)。将亚硝酸盐添加至肉块。使用绞肉机(X70型,HermannScharfen GmbH & Co,Witten,Germany)将肉和脂肪分别通过3mm孔板(orificeplate)绞碎,并且在4℃的冷藏温度放置过夜。
在MADO电动桌面切割机MTK 560(Pfeiffer GmbH,Dornhan,Germany)中将肉与三分之一的碎冰和全部磷酸盐混合。其后将剩余碎冰的一半(总量的三分之一)和盐与酶(磷脂酶A1(YieldMAX,Chr.Hansen,Denmark)和磷脂酶A2(Lecitase 10L,Novozymes Denmark,剂量(LEU/g脂肪)示于表3)一起添加。将剩余碎冰和干成分(香料、麦芽糊精和抗坏血酸盐)加入并且切碎(chop)直至混合物达到10℃的温度。将脂肪加入并且切碎直至混合物达到12-14℃的温度。
在终止混合后,在装罐之前在Komet Plus Vac 24(Komet MaschinenfabrikGmbH,Plochingen,Germany)中将肉乳化物抽真空9×15秒。然后将肉糊(meatbatter)填入罐中,在每个罐顶部留下5%顶部空间(head space)。在密封之前,另外将罐抽真空9次(vacuumed an additional 9 times)。使用Seaming V10Automat(Lanico,Germany)将罐密封,并且在100℃热水中加热95分钟。
表2.午餐肉的组成
成分 量(%)
猪肉5.3% 脂肪51.40%
碎冰 24.40%
猪油(pork fat) 21.83%
亚硝酸盐 1.00%
盐 0.50%
香料 0.50%
三聚磷酸钠 0.37%
将磷脂酰胆碱(PC)和磷脂酰乙醇胺(PE)的水解量分别测定为与对照样品相比PC含量和PE含量的百分比降低。通过HPLC用以下方法测定PC和PE:在30ml氯仿/甲醇(2∶1体积/体积)中提取均质化午餐肉样品的脂质级分并且通过在4℃在3000rpm离心30分钟来将其分离。将带有脂质的有机溶剂相定量转移至蒸发烧瓶并且在45-48℃干燥。在脂质类分离(lipid class separation)之前,将全部脂质提取物再溶解在2.5ml氯仿中。通过添加2×2ml己烷来清洗固相提取NH2柱(BondElut NH2,Varian Incorporated,CA,USA)并且将其用真空排空。然后将样品添加至柱并且用真空引导其通过。为了去除中性脂质,添加2×2ml氯仿/2-丙醇(2∶1体积/体积)。通过添加2×2ml 2%乙酸的醚溶液来去除游离脂肪酸。向柱添加甲醇(2×2ml)并且将含有(溶血-)磷脂的脂质级分收集在玻璃杯中并且用氮气使其蒸发(evaporated with nitrogen)。
为梯度驱动分离(gradient-driven separation)制备两个洗提剂。洗提剂A由甲醇、乙酸、三乙胺和己烷的混合物(897∶18∶15∶70体积/体积/体积/体积)组成,而洗提剂B是丙酮、乙酸、三乙胺和己烷的混合物(897∶18∶15∶70体积/体积/体积/体积)。
制备混合的标准样品,其含有1425微升洗提剂B,和分别为37.5微升的合成磷脂酰乙醇胺和磷脂酰胆碱。
使用Dionex UCI-50高效液相层析(Dionex Softron GmbH,Germering,Germany)来分离和定量各个(溶血-)磷脂种类。所述层析包含ASI-100自动样品注射器(Dionex,Germering,Germany)和P680 HPLC泵(Dionex,Germering,Germany),键合的二氧化硅柱(bonded silica column)(100 Diol,5微米,序列号415614,Merck KgaA,Darmstadt,Germany),和Alltech ELSD 2000蒸发式光散射检测仪(Alltech Associates Inc.,IL,USA)。
通过用直径3.5mm的平端圆柱型不锈钢冲杆(plunger)的穿透力来测量质地。将冲杆连在与Instron 4301机(Instron Limited,Buck-inghamshire,England)的十字头(crosshead)连接的100N静载荷元件上。将十字头速度运行在50mm/分钟,并且使用Series IX Software(M12-13984-EN,Instron Limited,Buckinghamshire,England)记录力-距离变形曲线,并且测定最大穿透力。在测试的每个午餐肉罐中进行三次穿透。分别在8℃和23℃对样品进行测试。将对每个样品测得的平均最大穿透力归纳在表3中。
表3.酶类型和剂量(LEU/g脂肪),磷脂酰胆碱(PC)和磷脂酰乙醇胺(PE)的水解程度(%),和最终肉样品中在8℃和23℃的最大穿透力(N)
Claims (11)
1.用于制造基于肉的食品的方法,其包括:
a)将肉与磷脂酶A1或磷脂酶A2接触;
b)加热经磷脂酶处理的肉;和
c)由经磷脂酶处理的肉制造食品;
其中步骤b)在步骤c)之前、期间或之后进行。
2.权利要求1的方法,其中将所述经上述的磷脂酶处理的肉加热至足以使磷脂酶失活的温度并且持续足以使磷脂酶失活的时间。
3.权利要求1的方法,其中将所述经上述的磷脂酶处理的肉加热到至少50℃。
4.权利要求3的方法,其中将所述经上述的磷脂酶处理的肉加热至50-140℃的温度。
5.前述权利要求中任一项的方法,其中所述待与上述的磷脂酶接触的肉是绞碎的肉。
6.前述权利要求中任一项的方法,其中所述食品是乳化肉制品。
7.前述权利要求中任一项的方法,其中所述食品包含至少30%的肉。
8.前述权利要求中任一项的方法,其中所述磷脂酶是纯化的磷脂酶。
9.前述权利要求中任一项的方法,其中经上述的磷脂酶处理的肉的磷脂已被修饰,所述修饰通过水解磷脂酰胆碱加磷脂酰乙醇胺总量的至少5%来进行。
10.前述权利要求中任一项的方法,其中经磷脂酶处理的肉的磷脂已被修饰,所述修饰通过水解磷脂酰胆碱加磷脂酰乙醇胺总量的至少20%来进行。
11.通过权利要求1-10中任一项的方法可获得的基于肉的食品。
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CN1638647A (zh) * | 2002-02-20 | 2005-07-13 | 诺维信公司 | 制备干酪的方法 |
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WO2007051472A1 (en) | 2007-05-10 |
DK1945048T3 (da) | 2010-07-26 |
NZ567239A (en) | 2010-08-27 |
BRPI0618009A2 (pt) | 2011-08-16 |
US20090011086A1 (en) | 2009-01-08 |
ATE462310T1 (de) | 2010-04-15 |
EP1945048B1 (en) | 2010-03-31 |
AU2006310877B2 (en) | 2012-03-22 |
PL1945048T3 (pl) | 2010-09-30 |
EP1945048A1 (en) | 2008-07-23 |
AR056168A1 (es) | 2007-09-19 |
AU2006310877A1 (en) | 2007-05-10 |
US20110142992A1 (en) | 2011-06-16 |
ES2343587T3 (es) | 2010-08-04 |
CN101296626A (zh) | 2008-10-29 |
DE602006013341D1 (de) | 2010-05-12 |
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