CN101039596A - 含有疏水蛋白的充气食品 - Google Patents
含有疏水蛋白的充气食品 Download PDFInfo
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- CN101039596A CN101039596A CNA2005800322108A CN200580032210A CN101039596A CN 101039596 A CN101039596 A CN 101039596A CN A2005800322108 A CNA2005800322108 A CN A2005800322108A CN 200580032210 A CN200580032210 A CN 200580032210A CN 101039596 A CN101039596 A CN 101039596A
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- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
Abstract
提供了包含疏水蛋白的冷冻组合物。还提供了疏水蛋白在抑制冷冻食品中冰晶生长和/或改变冰晶习性的用途。
Description
发明领域
本发明涉及包含疏水蛋白的冷冻产品。
发明背景
冷冻产品在贮藏过程中,其中存在的冰晶因动力学过程如重结晶其大小往往会增大。这会导致不良的产品特性,如不良的外观和不可接受的口感,和/或导致产品损害。之前已有建议使用称为“抗冻蛋白”(也称“制约冰晶结构的蛋白质(ice structuring protein)”)的蛋白质来抑制冰重结晶过程。
发明概述
我们发现,在真菌中发现的一类称为疏水蛋白的蛋白质也能够抑制冷冻产品中的冰晶生长。
因此,本发明提供冷冻组合物,如冷冻食品,所述组合物包含疏水蛋白,优选分离形式的疏水蛋白。在相关的方面,本发明提供冷冻组合物如冷冻食品,其包含能够在空气-液体表面装配的形式的疏水蛋白,并提供加入了所述形式的疏水蛋白的冷冻组合物,如冷冻食品。
优选疏水蛋白是II类疏水蛋白。
在优选的实施方案中,疏水蛋白的含量是至少0.001wt%,更优选至少0.01wt%。
在一个实施方案中,冷冻组合物是充气的。在另一个实施方案中,冷冻组合物是不充气的。
在相关的方面,本发明提供用以生产本发明的冷冻食品的组合物,所述组合物包含疏水蛋白,优选分离形式的疏水蛋白,以及至少一种所述食品的其余成分,所述组合物为非冷冻形式。优选所述组合物包含所述食品的所有其余成分。
在相关的实施方案中,本发明提供用以生产本发明的冷冻食品的干组合物,所述组合物包含疏水蛋白,优选分离形式的疏水蛋白,以及至少一种所述食品其余的非液体成分。优选所述组合物包含所述食品所有其余的非液体成分。
本发明还提供疏水蛋白在抑制冷冻组合物中冰晶生长的方法中的用途。优选疏水蛋白用以抑制冰重结晶。
在相关的方面,本发明还提供疏水蛋白在改变冷冻组合物中冰晶习性的方法中的用途。
本发明进一步提供抑制冷冻组合物中冰晶生长例如冰重结晶的方法,所述方法包括在所述组合物冷冻之前和/或冷冻过程中向组合物加入疏水蛋白。
在相关的方面,本发明提供改变冷冻组合物中冰晶习性的方法,所述方法包括在所述产品冷冻之前和/或冷冻过程中向组合物加入疏水蛋白。
在上述用途和方法的优选实施方案中,所述组合物是冷冻食品。
发明详述
除非另有定义,本文使用的所有技术和科学术语具有相关领域(如冷却糖食品/冷冻糖食品制造、化学和生物技术领域)普通技术人员通常理解的相同含义。冷却/冷冻糖食品制造中使用的各种术语和技术的定义和描述见于Ice Cream,第4版,Arbuckle(1986),VanNostrand Reinhold Company,New York,NY。用于分子和生化方法技术可见于Sambrook等,Molecular Cloning:A Laboratory Manual,第3版(2001)Cold Spring Harbor Laboratory Press,Cold Spring Harbor,N.Y.和Ausubel等,Short Protocols in Molecular Biology(1999)第4版,JohnWiley & Sons,Inc.,完全版本名为Current Protocols in MolecularBiology)。
疏水蛋白
疏水蛋白是定义明确的一类蛋白质(Wessels,1997,Adv.Microb.Physio.38:1-45;Wosten,2001,Annu Rev.Microbiol.55:625-646),能够在疏水/亲水界面自装配,具有以下保守序列:
Xn-C-X5-9-C-C-X11-39-C-X8-23-C-X5-9-C-C-X6-18-C-Xm(SEQ ID No.1)
其中X代表任何氨基酸,n和m独立代表整数。通常,疏水蛋白的长度最多达125个氨基酸。保守序列中的半胱氨酸残基(C)是二硫键的一部分。在本发明情形中,术语疏水蛋白具有更宽的含义,包括仍显示在疏水-亲水界面自装配产生蛋白质膜的特性的功能等价蛋白质,如包含以下序列或其部分、仍显示在疏水-亲水界面自装配产生蛋白质膜的特性的蛋白质:
Xn-C-X1-50-C-X0-5-C-X1-100-C-X1-100-C-X1-50-C-X0-5-C-X1-50-C-Xm(SEQ ID No.2)
按照本发明的定义,自装配可通过将蛋白质吸附到特氟隆(Teflon)上,并用圆二色性确定二级结构(一般是α-螺旋)的存在来检测(De Vocht等,1998,Biophys.J.74:2059-68)。
膜的形成可通过将特氟隆薄片(sheet)在蛋白质溶液中温育,然后用水或缓冲液至少洗涤三次来确定(Wosten等,1994,Embo.J.13:5848-54)。蛋白质膜可用任何合适的方法来显现,如用荧光标志进行标记,或者使用荧光抗体,这在本领域是已经确立的。m和n的数值通常在0-2000的范围,但更通常是m和n加起来小于100或200。在本发明情形中,疏水蛋白的定义包括疏水蛋白和另一种多肽的融合蛋白以及疏水蛋白与其它分子如多糖的缀合物。
目前为止得到鉴定的疏水蛋白一般可归类为I类或II类疏水蛋白。这两种类型的疏水蛋白在真菌中已被鉴定为分泌蛋白,可在疏水-亲水界面自装配成两性膜。I类疏水蛋白的装配物相对不可溶,而II类疏水蛋白的装配物则容易溶解于各种溶剂中。
还在丝状细菌如放线菌(Actinomycete sp.)和链霉菌(Steptomycessp.)中鉴定出疏水蛋白样蛋白质(WO01/74864)。这些细菌蛋白质与真菌疏水蛋白相比,只能最多形成一个二硫键,因为它们只有两个半胱氨酸残基。这种蛋白质是具有SEQ ID No.1和2所示共有序列的疏水蛋白功能等价物的实例,落入本发明的范围之内。
可用任何合适的方法从天然来源如丝状真菌提取获得疏水蛋白。例如,可通过培养能将疏水蛋白分泌到生长培养基中的丝状真菌,或者通过用60%乙醇从真菌菌丝体提取,来获得疏水蛋白。特别优选从能天然分泌疏水蛋白的宿主生物分离疏水蛋白。优选的宿主是丝孢菌(例如木霉属)、担子菌和子囊菌。特别优选的宿主是食品级生物,如能分泌称为cryparin的疏水蛋白的栗疫病菌(Cryphonectriaparasitica)(MacCabe和Van Alfen,1999,App.Environ.Microbiol 65:5431-5435)。
或者,可用重组技术获得疏水蛋白。例如可对宿主细胞(通常是微生物)进行修饰以表达疏水蛋白,然后疏水蛋白可根据本发明进行分离和使用。用以将编码疏水蛋白的核酸构建物引入到宿主细胞中的方法是本领域公知的。已经从16种以上的真菌克隆了超过34个编码疏水蛋白的基因(参见例如WO96/41882,该专利给出了在双孢蘑菇(Agaricus bisporus)中鉴定出的疏水蛋白的序列;和Wosten,2001,Annu Rev.Microbiol.55:625-646)。也可以用重组技术来修饰疏水蛋白序列或者合成具有需要的/改进特性的新型疏水蛋白。
通常,用编码所需疏水蛋白的核酸构建物转化适当的宿主细胞或生物。可将编码所述多肽的核苷酸序列插入到编码转录和翻译的必需元件的合适表达载体中,插入的方式要使得所述核苷酸序列在适当条件下会被表达(例如在适当的方向上和正确的读框中,有适合的靶向和表达序列)。构建这些表达载体所需的方法是本领域技术人员公知的。
有多种表达系统可用来表达多肽编码序列。这些表达系统包括但不限于细菌、真菌(包括酵母)、昆虫细胞系统、植物细胞培养系统和植物,它们都用用适当的表达载体转化。优选的宿主是被认为是食品级——公认为安全(GRAS)的宿主。
合适的真菌包括酵母如(但不限于)酵母菌属(Saccharomyces)、(Kluyveromyces)、毕赤氏酵母属(Pichia)、汉逊酵母属(Hansenula)、假丝酵母属(Candida)、裂殖酵母属(Schizosaccharomyces)等属酵母和丝状真菌如(但不限于)曲霉属(Aspergillus)、木霉属(Trichoderma)、毛霉属(Mucor)、链孢霉属(Neurospora)、镰刀菌属(Fusarium)等属丝状真菌。
编码疏水蛋白的序列优选在氨基酸水平上与在自然界鉴定的疏水蛋白有至少80%的同一性,更优选有至少95%或100%的同一性。但是,本领域技术人员可作出不降低疏水蛋白的生物活性的保守置换或其它氨基酸变化。对于本发明的目的,这些拥有与天然疏水蛋白的高水平同一性的疏水蛋白,也包括在术语“疏水蛋白”当中。
疏水蛋白可例如按WO 01/57076描述的程序从培养基或细胞提取物中纯化,所述程序涉及将含疏水蛋白的溶液中存在的疏水蛋白吸附到表面上,然后将该表面与表面活性剂如Tween 20接触,以从该表面洗脱出疏水蛋白。另参见Collen等,2002,Biochim Biophys Acta.1569:139-50;Calonje等,2002,Can.J.Microbiol.48:1030-4;Askolin等,2001,Appl Microbiol Biotechnol.57:124-30和De Vries等,1999,Eur J Biochem.262:377-85。
冷冻组合物
冷冻组合物/冷冻产品包括冷冻食品和冷冻生物材料。冷冻食品包括冷冻植物来源材料如水果和蔬菜,冷冻动物来源材料如冷冻肉和鱼,以及冷冻加工食品如现成膳食、调味汁和冷冻糖食如冰淇淋、乳冰、冷冻酸乳酪、冰糕、糖浆饮料、冷冻蛋奶甜羹、水冻冰块、清凉果汁饮料、格兰尼它冰糕(Granitas)和冷冻果泥。
本发明的冷冻组合物可以是充气的或不充气的。术语“充气”指已例如通过机械手段有意地将气体引入到了产品中。气体优选是任何食品级气体,如空气、氮气或二氧化碳。充气程度,尤其是在充气食品的情形中,通常以“膨胀度”来定义。在本发明的情形中,%膨胀度按体积定义为:
((充气终产品的体积-混合料的体积)/混合料的体积)×100
产品的膨胀度须根据所需的产品特性而变。例如,冰淇淋的膨胀度水平通常是约70-100%,而水冻冰块则是25-30%。
不充气组合物如冷冻食品的膨胀度优选低于20%,更优选低于10%。不充气冷冻食品没有经历故意的处理步骤如搅打以增加气体含量。尽管如此,应认识到在不充气冷冻食品的制作过程中,可能有低水平的气体如空气掺入到其中。
冷冻糖食品
冷冻糖食包括通常包含乳或乳固体的糖食,如冰淇淋、乳冰、冷冻酸乳酪、冰糕和冷冻蛋奶甜羹,以及不含乳或乳固体的冷冻糖食,如水冻冰块、清凉果汁饮料、格兰尼它冰糕和冷冻果泥。
冷冻糖食可为复合产品的形式,产品中至少一部分或区域(如芯或覆层)不含有疏水蛋白。其实例是以下产品,其中冰淇淋芯没有疏水蛋白,包覆芯的冰淇淋、乳冰或水冻冰块覆层则含有疏水蛋白。应认识到,在复合产品的情况下,所加入的疏水蛋白的wt%只针对糖食中含有疏水蛋白的成分而不是针对整个产品来计算。
充气冷冻糖食的膨胀度优选是25%-300%,如25%-150%,更优选50-150%。
本发明冷冻组合物中存在的疏水蛋白的量一般须根据产品配方而变,在充气产品的情况下根据空气相的体积而变。通常,产品须含有至少0.001wt%疏水蛋白,更优选至少0.005或0.01wt%。通常产品须含有低于1wt%疏水蛋白。疏水蛋白可来自单一来源或多个来源,例如疏水蛋白可以是两种或多种不同的疏水蛋白多肽的混合物。
优选疏水蛋白是II型疏水蛋白。
本发明还涵括用以生产本发明的冷冻食品的组合物,所述组合物包含疏水蛋白。一般地,疏水蛋白须为分离形式,通常至少部分纯化,如以固形物重量计至少10%或20%纯。因此,疏水蛋白并不是作为天然表达疏水蛋白的天然生物如蘑菇的一部分来加入。相反,疏水蛋白通常须是从天然来源提取得到的,或者是通过在宿主生物中重组表达获得的。
这种组合物包括液体预混合料,例如用以生产冷冻糖食品的预混合料,和干混合料例如粉末料,其在冷冻之前或冷冻过程中加入含水液体如乳或水。
用以生产本发明的冷冻食品的组合物除包含疏水蛋白外,还须包含食品中通常包括的其它成分,例如糖、脂肪、乳化剂、调味剂等。所述组合物可包含制作所述食品所需的所有其余成分,使得它可容易地加工形成本发明的冷冻食品。
用以生产本发明的冷冻食品的干组合物除包含疏水蛋白外,还须包含食品中通常包括的其它成分,例如糖、脂肪、乳化剂、调味剂等。所述组合物可包含制作所述食品所需的所有其余非液体成分,使得使用者所要做的只是加入含水液体如水或乳,所述组合物就可容易地加工形成本发明的冷冻食品。这些干组合物的实例包括粉末料和颗粒料,可被设计成供工业使用和零售使用,且得益于体积减少和货架期延长。
向组合物中加入的疏水蛋白的形式和数量,要使得疏水蛋白能抑制冰晶生长如冰重结晶和/或改变冰晶习性。术语“加入”我们用以指有意地将疏水蛋白引入到组合物中,目的是利用它的抑制冰晶生长和/或改变冰晶习性的能力。因此,对于存在或加入了含有真菌污染物的成分,而真菌污染物可能含有疏水蛋白多肽的情况,这并不等同于本发明情形当中的加入疏水蛋白。
通常,加入到产品中的疏水蛋白的形式是能够在空气-液体表面自装配的形式。
通常,疏水蛋白以分离形式加入的本发明的组合物中,通常至少部分纯化,如固形物重量计至少10%纯。所谓“以分离形式加入”我们指疏水蛋白并不是作为天然表达疏水蛋白的天然生物如蘑菇的一部分来加入。相反,疏水蛋白通常须是从天然来源提取得到的,或者是通过在宿主生物中重组表达获得的。
所加入的疏水蛋白可用以减少或抑制冰晶的生长,例如以抑制冰重结晶的过程,和/或改变冰晶习性(即冰晶形状)。抑制和/或改变可在冷冻过程中和/或冷冻之后如贮藏过程中发生。在冷冻过程中对冰晶生长和/或冰晶习性的抑制可用来改变产品的质地。冰重结晶的抑制可提高产品对热滥用的稳定性。
疏水蛋白也可用来抑制细胞生物材料中的冰晶生长如冰重结晶和/或冰晶习性。这会有助于减少用以保存生物材料的冷冻方法对细胞造成的损害。这种生物材料包括单细胞生物和细胞系的培养物;配子如精子和卵子;和衍生自多细胞生物(动植物)的组织和器官。
因此,本发明还提供冷冻的细胞生物材料,其包含分离形式的疏水蛋白,优选包含至少0.001wt%疏水蛋白,条件是将人排除在外。
本发明进一步提供疏水蛋白在冷冻细胞生物材料中抑制冰晶生长如冰重结晶和/或改变冰晶习性的用途。抑制/改变可在生物材料的冷冻过程中和/或冷冻之后发生。
现参考以下只是示例性而非限制性的实施例对本发明作进一步的描述。
附图简述
图1是显示充气冷冻产品的剪切方案(regime)的示图。
图2是充气冷冻产品微结构的扫描电子显微照片-新鲜的和热滥用后的(放大倍数×100)。
图3是充气冷冻产品微结构的扫描电子显微照片-新鲜的和热滥用后的(放大倍数×300)。
图4显示不充气产品的扫描电子显微照片(无HFBII)(50×放大倍数)。
图5显示不充气产品的扫描电子显微照片(有HFBII)(50×放大倍数)。
实施例1-充气冷冻产品
用3种类型的蛋白质制备充气冷冻产品:
A:酪蛋白酸钠(Na Cas)
B:脱脂奶粉(SMP)
C:来自里氏木霉(Trichoderma reesei)疏水蛋白(HFB II)
对各产品在温度滥用方案之前和之后的微结构特性和物理特性进行比较。
材料
所用材料的具体细节在表1中总结,用以制备每种充气冷冻产品的配方在表2中显示。
表1.所用材料
成分 | 组成 | 供应商 |
酪蛋白酸钠 | 88-90%蛋白质,1.5%脂肪,6%水分 | DMV International,荷兰 |
脱脂奶粉 | 33-36%蛋白质,0.8%脂肪,3.7%水分 | United Milk,英国 |
II型疏水蛋白(HFB II) | 基本按WO00/58342和Linder等,2001,Biomacromolecules2:511-517的描述从里氏木霉纯化得到 | VTT Biotechnology,芬兰 |
精制椰子油 | Van den Bergh Foods,Limited | |
蔗糖 | Tate and Lyle,英国 |
表2.所用配方
混合料A | 混合料B | 混合料C | |
成分 | 浓度/wt% | ||
酪蛋白酸钠 | 2.0 | -- | -- |
脱脂如奶粉 | -- | 11.42 | -- |
HFB II | -- | -- | 0.2 |
椰子油 | 5.0 | 5.0 | 5.0 |
蔗糖 | 25.0 | 20.0 | 25.0 |
水 | 68.0 | 63.58 | 69.8 |
充气冷冻产品的制备
混合料(乳状液)制备
所有的混合料均按100g批量制备。对于混合料A和B(分别含有酪蛋白酸钠和脱脂奶粉),将蛋白质与蔗糖混合,用磁力搅拌器分散到冷水中。然后将溶液搅拌加热至60℃,保持5分钟,接着冷却至40℃。然后加入熔化的椰子脂,立即用Branson Sonifer(带6.4mm锥形探头)在70%振幅下将含水混合料超声处理3分钟,探头浸没在溶液中。然后将所得乳状液在-10℃水浴中快速冷却,直到溶液温度为5℃,以使脂肪滴结晶。将混合料在5℃下贮藏备用。
对于混合料C(含有HFB II),首先将蔗糖搅拌分散到冷水中。然后,将一半所需浓度的HFB II作为等分试样加入。接着将溶液在超声浴中温和超声处理30秒,以使HFB II完全分散。然后将此溶液在磁力搅拌器上搅拌和加热至40℃。在加入熔化的脂肪之前,再将溶液在超声浴中超声处理30秒。然后加入熔化的脂肪,所得混合料按对混合料A和B的描述进行乳化和冷却。接着向此冷溶液中加入另一HFB II等分试样,以使HFB II浓度达到0.2%。第一个0.1%HFBII用以使脂肪乳化和稳定化。HFB II的第二次加入则会提供足够过量的HFB II,以提供良好的充气作用和泡沫稳定性。
用Malvern Mastersizer 2000对冷冻的乳状液进行颗粒大小分析。
乳状液的分析
按照这一方法,我们能够制备出具有小脂肪滴的乳状液。表3总结了所测出的油滴大小。
表3.用Malvern Mastersizer 2000测出的乳状液颗粒大小
混合料 | 脂肪滴直径 |
D(3,2)/μm | |
A(Na Cas) | 0.4 |
B(SMP) | 0.25 |
C(HFB II) | 1.88 |
剪切冷冻方法
将80ml混合料在圆柱形垂直安装带夹套的不锈钢容器中同时进行剪切和冷冻,该容器的内部大小是高度105mm,直径72mm。该容器的盖占满其内部体积的54%,只留下46%(180ml)给样品。用以剪切样品的转子由正确大小的矩形推进器(尺寸72mm×41.5mm)组成,以在容器旋转过程中刮擦容器的表面边界(surface edge)。转子上还连接有两个半圆形(60mm直径)高剪切叶片,该叶片与该矩形附加装置成45°角定位。容器外包围着夹套,夹套里有冷却剂流过。
充气和冷冻样品(prototype)大体上如下产生:用推进器在高剪切速度下混合密闭容器里的混合料,以掺入空气。同时,冷却剂流过容器夹套,使混合料冷冻下来。推进器还刮擦容器内壁,将内壁上形成的冰除去并掺入到混合料当中。开始时用高剪切使混合料充气,然后将剪切速度降下来,以便更好地进行冷冻。每种混合料所用的剪切方案在图1中图示。
对于混合料A和B(分别含有酪蛋白酸钠和脱脂奶粉)的冷冻和充气步骤,设定冷冻剂(设定在-18℃)从时间=0分钟开始循环。混合料A和B在开始时搅拌速度较慢,是为了便于冷却混合料和产生一定的粘度(通过冰的形成和掺入),然后才用较高的剪切速度进行充气。在高速度下短时间就将空气掺入,然后逐步降低速度,以让样品达到至少-5℃。
对于混合料C(含有HFB II),将容器冷却至约5℃,加入样品,启动高剪切充气。冷却剂(设定在-18℃)在第9分钟才开启进行循环,因为要产生100%膨胀度所需的时间增加。一旦开启冷却剂进行循环(第9分钟),则采取和前面的A和B一样的剪切-冷却模式。
在整个过程结束时,测量膨胀度,将样品(大约15g)放入小罐中。将每种产品在设定在-80℃的冷冻装置中再冷却10分钟,然后在-20℃下贮藏。
充气冷冻产品的分析
所有的充气冷冻产品均按两种温度方案进行贮藏:
(a)-20℃。随后在生产后一星期内进行分析,我们认为这种产品是“新鲜”产品。
(b)使温度滥用样品经历-10℃贮藏1或2个星期,然后分析前在-20℃下贮藏。
表4.从混合料A、B和C制备的产品的过程细节和产品膨胀度。
样品 | 1200rpm下剪切时间 | 膨胀度 | 终产品温度 |
min | % | ℃ | |
A(Na Cas) | 1 | 103 | -5.3 |
B(SMP) | 1 | 98 | -8 |
B(SMP) | 1 | 94 | -5.6 |
C(HFB II) | 10 | 75 | -5 |
终产品作如下分析:
刚制得的产品的膨胀度
新鲜产品和温度滥用产品的SEM分析
新鲜产品和温度滥用产品的熔化行为
膨胀度
每种产品的膨胀度在表4中总结。所有的混合料都是可充气的,掺入了大量的空气。
微结构稳定性:方法
扫描电镜术(SEM)
每种产品的微结构用低温扫描电镜术(LTSEM)来显现。将样品在干冰上冷却至-80℃,切出样品断片。用Tissue Tek:OCTTM化合物(PVA 11%、Carbowax 5%和85%非反应性成分),将大小约5mm×5mm×10mm的此断片安放在样品支持器上。将样品包括支持架投入液氮浆(slush)中,并转移到低温制备室Oxford Instrument CT1500HF中。制备室处在约10-4巴真空下,样品升温至-90℃。冰被慢慢蚀刻,暴露出不由冰本身产生的表面细节,因此在此温度下要在恒定真空下除水60-90秒。样品一旦蚀刻好,就将其冷却至-110℃以结束升华作用,并使用氩等离子体包覆以金。这个过程也发生在真空下,施加10-1毫巴压力、6毫安电流45秒。然后将样品转移到常规的扫描电子显微镜(JSM 5600),该扫描电子显微镜装配有温度-160℃的Oxford Instruments冷台。检查样品,并通过数字影像撷取软件捕获目的区域。
冷冻断裂透射电镜术(TEM)
将样品放在置于干冰床上的铝盘上,用冷的解剖刀切出大约5mm×3mm×3mm的小块。使用Tissue-Tek O.C.T化合物(Sakura Finetek,Europe BV),将样品块垂直安放在大的“礼帽”冷冻断裂支持器中。立即在液氮下将该支持器放入Cressingtion CFE-50的转移装置中,并转移到冷冻断裂室(-180℃)中。样品用摆动式切片刀击打一次发生断裂,然后在-95℃下蚀刻10分钟。蚀刻出的表面用铂/碳旋转造影(rotary shadow)(45°)至2nm的厚度,然后用碳包覆至10nm的厚度。将包覆样品移出冷冻断裂室和转移装置,金属复制品就漂浮离开样品到水上。将复制品在铬酸中清洗,用水洗涤数次,然后收集在400目铜EM网格上。让网格干燥,然后进行TEM检查。
用在100KV下操作的JEOL 1200EX II显微镜进行TEM检查。用Bioscan照相机和Digital Micrograph软件(Gatan Inc)获取影像。
微结构分析:结果
用扫描电镜术(SEM)检查了新鲜的和温度滥用的冷冻产品的微结构。不同放大倍数的代表性影像见图2和3。
新鲜样品的结果显示,含有HFB II的产品(从混合料C制备)显著地且令人惊异地不同于含有更多常规蛋白质(即混合料A或B)的产品。所观察到的不同特性有:气胞更小,冰晶棱角更多和冰晶融合稍多。对于产品A和B,我们估计新鲜样品中冰晶大小为直径50-100μm。对于产品C,我们估计冰晶大小为直径40-60μm。
温度滥用后,SEM影像清楚显示,含有HFB II的产品(从混合料C制备)还保留着它原来的微结构,即相对来说冰晶显现和气泡粗化的情况极少。在-10℃下贮藏1个星期和2个星期也是这样。但是,含有Na Cas和SMP(分别来自混合料A和B)的样品仅仅在-10℃的温度滥用下一个星期后,就显示出非常严重的气泡和冰结构粗化现象。
总的来说,现清楚,被制成含HFBII的冷冻产品比用酪蛋白酸钠或脱脂奶粉制成的冷冻产品显示出对温度滥用高得多的稳定性。HFBII对气泡和冰晶大小和稳定性两方面都有影响。
熔化行为:方法
将冷冻产品的样品放在室温(20℃)下的金属网格上。观察产品随时间的熔化情况,特别是形状保持力和泡沫稳定性。
熔化行为:结果
这些微结构上的差异(稳定的泡沫和稳定的冰)对冷冻产品的熔化行为有一定的影响。从混合料C(含有HFBII)制得的充气冷冻样品,比起用酪蛋白酸钠或脱脂奶粉(即分别混合料A和B)制得的产品,在熔化时能更好地保持其形状。
随着冰发生熔化形成水,水会流过金属网格。但是,对于含HFBII的产品,许多泡沫仍保留在网格上,网格下面可观察到少数稳定的泡沫滴(数据未给出),这两个特性在常规的蛋白质(酪蛋白酸钠和脱脂奶粉)上均没有观察到。这说明每种所用的蛋白质之间在泡沫稳定性方面的差异。
产品A、B和C之间的质地差异
三种样品在-10℃下贮藏一个星期后(即稳定滥用样品),也可观察到它们之间在质地方面有明显的差异。在触摸用酪蛋白酸钠(A)和脱脂奶粉(B)制得的产品时,注意到这些产品具有非常松软和非常易成薄片的质地,难以从用于衬垫样品罐的涂硅纸干净地取下来。另一方面,用HFB II制得的产品(C)非常结实,能非常干净地从衬垫样品罐的涂硅纸释放出来。换句话说,用HFB II制备的产品比用酪蛋白酸钠或脱脂奶粉制备的产品,在微观和宏观规模上对温度滥用都显示出高得多的稳定性。
实施例2:非充气冷冻产品
制备了两种溶液,一种含有来自里氏木霉的疏水蛋白HFB II。另一种不含疏水蛋白。两种溶液的组成在表5中显示。
表5-非充气产品的配方
样品1 | 样品2 | |
成分 | 浓度/wt% | |
HFB II | 0 | 0.1 |
黄原胶 | 0.5 | 0.5 |
蔗糖 | 25.0 | 25.0 |
水 | 74.5 | 74.4 |
HFB II由上述VTT Biotechnology提供,蔗糖由Tate & Lyle提供。黄原胶由CP Kelco(美国亚特兰大)提供,属冷水可分散级(KeltrolRD)。
非充气冷冻产品的制备和分析
两种溶液均按100g批量制备。如下制备蔗糖/黄原胶溶液:将所需量的室温去离子水加入到蔗糖和黄原胶的干混合物中,然后用磁力搅拌器混合至溶质完全溶解。在样品2的情况下,然后将HFB II以5.3mg/ml溶液的等分试样加入,接着将溶液在磁力搅拌器上再混合10分钟。
非充气溶液的冷冻以静止方式进行(即不同时施加剪切)。用每种溶液充满8ml体积的小平皿。然后将平皿放入-18℃家用冷藏橱柜中24小时,在这个时间内样品发生冰冻。
冰冻后,用实施例1所述的相同制备方法,通过SEM分析每种样品的微结构。
微结构分析-结果
每种样品50×放大倍数的代表性SEM影像可见于图4和5。可辨别出,含有HFB II的溶液(样品2)的微结构更细,含有特征尺寸更小的冰晶。例如,样品1(图4)的伸长的树枝状结构比样品2(图5)见到的更宽更长。这说明了疏水蛋白对减少冰晶生长过程——导致尺寸更小的冰晶的影响。
上文各单独节内容提及到的本发明各个不同的特征和实施方案,在适当的情况下作了必要的修正也适用于其它节。因此,在适当的情况下可将在一节中载明的特征与在其它节中载明的特征结合在一起。
以上说明书中提到的所有出版物通过引用结合到本文中。不背离本发明的范围对已描述的本发明方法和产品所作的各种修改和变化,对本领域技术人员来说将是显而易见的。虽然本发明已结合具体的优选实施方案进行了描述,但应认识到,要求保护的本发明不应不适当地限制于这些具体实施方案。实际上,对已描述的本发明各实施方式所作的各种对相关领域技术人员来说显而易见的修改,都意在落入以下权利要求的范围内。
序列表
<110>Unilever et al.
<120>冰冻食品
<130>F3383
<140>PCT/EP2005/006997
<141>2005-06-27
<150>EP04254483.3
<151>2004-07-27
<150>EP05251282.9
<151>2005-03-27
<160>2
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Claims (15)
1.一种冷冻组合物,所述组合物包含分离形式的疏水蛋白。
2.权利要求1的冷冻组合物,所述组合物包含至少0.001wt%的疏水蛋白。
3.一种冷冻组合物,所述组合物包含至少0.001wt%的疏水蛋白。
4.权利要求1-3中任一项的冷冻组合物,其中所述疏水蛋白是II型疏水蛋白。
5.权利要求1-3中任一项的冷冻组合物,所述组合物是不充气的。
6.前述权利要求中任一项的冷冻组合物,所述组合物是食品。
7.权利要求6的冷冻食品,所述食品是冷冻糖食品。
8.一种用以生产权利要求6或7的冷冻食品的组合物,所述组合物包含分离形式的疏水蛋白以及所述食品的其余成分,所述组合物是未冷冻形式。
9.一种用以生产权利要求6或7的冷冻食品的干组合物,所述组合物包含分离形式的疏水蛋白以及所述食品其余的非液体成分。
10.疏水蛋白在抑制冷冻组合物中冰晶生长的用途。
11.权利要求10的用途,其中所述冷冻组合物是食品。
12.疏水蛋白在改变冷冻组合物中冰晶习性的用途。
13.权利要求12的用途,其中所述冷冻组合物是食品。
14.疏水蛋白在改进冷冻组合物的温度耐受性中的用途。
15.权利要求14的用途,其中所述冷冻组合物是食品。
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Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101919428A (zh) * | 2009-06-02 | 2010-12-22 | 荷兰联合利华有限公司 | 充气的焙烤产品 |
CN102448313A (zh) * | 2009-05-29 | 2012-05-09 | 荷兰联合利华有限公司 | 水包油型乳液 |
CN102448312A (zh) * | 2009-05-29 | 2012-05-09 | 荷兰联合利华有限公司 | 水包油型乳液 |
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CN102448313A (zh) * | 2009-05-29 | 2012-05-09 | 荷兰联合利华有限公司 | 水包油型乳液 |
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CN101919428A (zh) * | 2009-06-02 | 2010-12-22 | 荷兰联合利华有限公司 | 充气的焙烤产品 |
CN103153086A (zh) * | 2010-10-20 | 2013-06-12 | 荷兰联合利华有限公司 | 包含疏水蛋白的发泡剂 |
CN103491799A (zh) * | 2011-02-02 | 2014-01-01 | 荷兰联合利华有限公司 | 充气食品 |
CN103491799B (zh) * | 2011-02-02 | 2015-04-29 | 荷兰联合利华有限公司 | 充气食品 |
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