CN104582508B - 制备血清蛋白质浓缩物的方法 - Google Patents
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- CN104582508B CN104582508B CN201380042937.9A CN201380042937A CN104582508B CN 104582508 B CN104582508 B CN 104582508B CN 201380042937 A CN201380042937 A CN 201380042937A CN 104582508 B CN104582508 B CN 104582508B
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Abstract
本发明涉及一种以乳清样本制备具有降低的乳糖含量的血清蛋白质浓缩物的方法和包含该浓缩物的食品,其中该浓缩物的特征在于其具有每千克蛋白质≤0.5克乳糖的浓度,该浓缩物可从该方法得到。
Description
技术领域
本发明涉及一种以乳清样本制备蛋白质浓缩物的方法和包含该浓缩物的产品,其中该浓缩物的特征在于其具有每千克蛋白质≤0.5克乳糖的浓度,该浓缩物可从该方法得到。
背景技术
乳清(whey)(或“乳清(lactoserum)”)表示乳的液体部分且除了蛋白质、矿物盐、痕量脂类和乳糖降解产物(乳酸、葡萄糖、半乳糖)之外还包含高百分含量(在50%至75%之间)的乳糖。乳清的蛋白质含量在约8%至14%范围内;特别地,蛋白质混合物的存在是可能的,蛋白质中的主要蛋白质为白蛋白、β-乳球蛋白、α-乳白蛋白、血清白蛋白、乳铁蛋白、免疫球蛋白;必需氨基酸类(亮氨酸、异亮氨酸、缬氨酸、苏氨酸、色氨酸、赖氨酸、苯丙氨酸等)。
各种研究证明了乳清蛋白质怎样对健康具有有益作用。特别地,Wong等报告了:由于乳清蛋白质中存在免疫球蛋白类,乳清蛋白质摄入增强了免疫功能(Wong&Watson1995.Immunomodulatory effects of dietary whey proteins in mice.J Dairy Res.62(2):359-68)。此外,还建议了乳清蛋白质摄入可怎样对对抗癌症发展具有预防作用(Hakkak等,Dietary whey protein protects against azoxymethane-induced colontumors in male rats.Cancer Epidemiol.Biomarkers Prev.10(5):555–8.PMID11352868)。
从医学观点看,特定兴趣不仅指向乳清免疫球蛋白类,还指向乳铁蛋白,乳铁蛋白对健康的有益作用(诸如改善免疫、抗菌、抗病毒、抗癌活性)已被描述。
乳清蛋白质还以其对皮肤的有益作用而著称。
即使如上所述,一系列有益作用与乳清蛋白质相关联,乳清蛋白质的摄入或使用通常被乳清蛋白质存在于其中的乳清的特征是高含量乳糖的事实限制。特别地,在世界人口中,对该分子的不耐症是普遍的;这主要是因为由于缺乏乳糖酶受试者无法代谢乳糖。该不耐症的发生因国家而显著改变,美国的成人人口中为约22%的百分比,南欧人口中达到约70%。
最普遍的乳糖不耐症相关的症状基本在于肠胃异常(诸如腹痛和腹部痉挛、肠水平上感觉肿胀和绷紧、蠕动增加、胃气胀、鼓胀、腹泻(diarrhoic bowel movement)等)。
最优异的乳糖不耐症的治疗为节食方案,其中富含乳糖食品(因此包括乳清)的贡献降低。然而,该治疗特别不适于不耐症受试者为例如其中乳清摄入是基本的以保证乳清蛋白质的适当分布的儿童受试者、营养不良的受试者、具有免疫缺陷的受试者等的情况。
因此,已知领域的情况感觉需要限定允许纯化来自乳清的血清蛋白质的混合物、使乳清中存在的乳糖的含量最小化和因此保证乳清中典型的蛋白质、氨基酸等的组成的保持的技术。
发明内容
本发明涉及一种以乳清(lactoserum)(也称为乳清(whey))制备蛋白质浓缩物的方法。特别地,该方法包括一系列操作步骤,这一系列操作步骤的最终结果是从乳清获得具有特别有利的特征的蛋白质(或也称为“血清蛋白质”)浓缩物。
实际上,本申请中描述的方法允许降低乳清中存在的乳糖的含量,并保持了血清蛋白质中的乳糖的含量,因此,换言之,没有改变或变更蛋白质、氨基酸、矿物盐等中的乳糖的特有组成。
特别地,由本申请描述的方法可获得的血清蛋白质浓缩物的特征在于其具有每千克蛋白质≤0.5克乳糖的浓度。此外,从根据以下描述的乳清分离的蛋白质具有至少96%的纯度。
因此,本说明书的目的的方法可有利地用于以乳清制备血清蛋白质浓缩物,该血清蛋白质浓缩物意欲用于例如具有乳糖不耐症或过敏症的受试者和/或需要血清蛋白质的重大贡献的受试者。
此外,本发明的作者还观察到,本申请描述的方法产生了特征是不含有难闻气味和味道、典型地代替乳清蛋白质的血清蛋白质浓缩物。最终分析中该另一优点决定了例如在随后阶段(诸如包含其的产物的制备)中该浓缩物的更容易的用途(通过示例但不限于)可为食品、化妆品、生物技术产品等。
因此,本发明的第一目的为一种以乳清样本制备具有降低的乳糖含量的血清蛋白质浓缩物的方法,该方法包括以下步骤:
a)浓缩该乳清样本,因此获得在约150克/升与300克/升之间的蛋白质浓度;
b)将该乳清浓缩物经历至少一个渗滤步骤;
c)稀释该渗滤的蛋白质浓缩物,因此获得蛋白质浓度在约50克/升至90克/升范围内的溶液;
d)向该溶液中加入每升溶液约5至10克的热解硅石;
e)将每千克蛋白质约70至300ml的乙醇(95%,体积/体积)加入该溶液;
f)将步骤e)获得的溶液的pH酸化为约4.5至5的值;
g)将酸化的溶液加热至约55℃与70℃之间的温度至少20分钟,其中该温度在约30分钟内达到;
h)将溶液冷却至约10℃与20℃之间的温度;
i)进行冷却的蛋白质溶液的分离;
l)将分离溶液的pH带至约5.8至6.8的值;
m)将步骤l)获得的溶液经历至少一个微孔过滤的过程,因此获得蛋白质浓度在约100克/升与300克/升之间的蛋白质浓缩物;
n)将蛋白质浓缩物经历至少一个渗滤步骤;
o)干燥和/或冷冻干燥该蛋白质浓缩物,
其中来自乳清的该血清蛋白质浓缩物的浓度为每千克蛋白质≤0.5克乳糖。
本说明书的第二目的为来自乳清的蛋白质(或血清蛋白质)浓缩物,每千克蛋白质包括≤0.5克乳糖。
本申请的描述的另外目的为包括以上乳清蛋白质浓缩物的食品、化妆品或生物技术产品。
具体实施方式
本发明涉及一种以乳清样本制备具有降低的乳糖含量的血清蛋白质浓缩物的方法。换言之,本申请描述的方法允许分离乳清中包含的蛋白质混合物,降低或消除乳清的起始样本中存在的乳糖。
本说明书中,术语“乳清(lactoserum)”或“乳清(whey)”是指从粗制凝乳酶分离之后的全脂乳、脱脂乳或半脱脂乳获得的液体部分。
特别地,本说明书的目的的方法包括以下操作步骤:
a)浓缩该乳清样本,因此获得在约150克/升与300克/升之间的蛋白质浓度;
b)将该乳清浓缩物经历至少一个渗滤步骤;
c)稀释该渗滤的蛋白质浓缩物,因此获得蛋白质浓度在约50克/升至90克/升范围内的溶液;
d)向该溶液中加入每升溶液约5至10克的热解硅石;
e)将每千克蛋白质约70至300ml的乙醇(95%,体积/体积)加入该溶液;
f)将步骤e)获得的溶液的pH酸化为约4.5至5.0的值;
g)将酸化的溶液加热至约55℃与70℃之间的温度至少20分钟,其中该温度在约30分钟内达到;
h)将溶液冷却至约10℃与20℃之间的温度;
i)进行冷却的蛋白质溶液的分离;
l)将分离溶液的pH带至约5.8至6.8的值;
m)将步骤l)获得的溶液经历至少一个微孔过滤的过程,因此获得蛋白质浓度在约100克/升与300克/升之间的蛋白质浓缩物;
n)将蛋白质浓缩物经历至少一个渗滤步骤;
o)干燥和/或冷冻干燥该蛋白质浓缩物,
其中来自乳清的该血清蛋白质浓缩物的浓度为每千克蛋白质≤0.5克乳糖。
根据本申请描述的方法的蛋白质浓缩物的制备可以以可包括甜乳清和酸乳清或可包含仅甜乳清或仅酸乳清的乳清样本为原料来进行。对本发明的目的有用的乳清可为任意来源;因此仅通过非限制性示例,该乳清可从人乳、牛乳或羊乳等获得。
如上所示,该方法包括浓缩乳清起始样本的步骤,该步骤可根据本领域技术人员已知且适于该目的的任意一种技术来进行。在该方法的优选实施例中,浓缩步骤通过超滤来进行。例如,根据上示步骤a),超滤通过使用截留(cut-off)为约10000道尔顿与15000道尔顿之间的薄膜来进行。因此,通过说明,可使用截留为10000、11000、12000、13000、14000和/或15000的薄膜。为了获得蛋白质浓度为至少150克/升的乳清的目的,会需要一个或多个超滤步骤和/或使用一个或多个以上说明的薄膜。
下一阶段(即以上方法的步骤b))在于降低根据上述的乳清浓缩物中乳糖的最大的可能的量。乳糖含量的全部消除或部分降低可例如通过至少一个渗滤步骤来进行。“渗滤”一般是指通过进行连续加入溶剂的超滤将微溶质与分子(本情况下为蛋白质)的溶液分离。例如,根据本发明的方法的渗滤可通过使用去矿物质水、蒸馏水和/或渗透水而不是盐水溶液作为溶剂来进行。仅通过示例,每体积乳清浓缩物可使用三体积去矿物质水。在本发明的一个实施例中,乳清浓缩物的渗滤步骤为至少3次且每体积乳清浓缩物使用三体积去矿物质水来进行。
然后将由于以上与起始乳清样本相比特征是降低的乳糖含量的渗滤的乳清通过加入溶剂稀释,因此获得蛋白质含量为约50克/升至90克/升的渗滤的乳清的溶液。特别地,该溶液的蛋白质含量可以为约50克/升、51克/升、52克/升、53克/升、54克/升、55克/升、56克/升、57克/升、58克/升、59克/升、60克/升、61克/升、62克/升、63克/升、64克/升、65克/升、66克/升、67克/升、68克/升、69克/升、70克/升、71克/升、72克/升、73克/升、74克/升、75克/升、76克/升、77克/升、78克/升、79克/升、80克/升、81克/升、82克/升、83克/升、84克/升、85克/升、86克/升、87克/升、88克/升、89克/升、90克/升。在本发明的一个实施例中,渗滤的乳清的溶液的蛋白质浓度为约70克/升。例如,渗滤的乳清的溶液的稀释通过使用去矿物质水作为溶剂来进行。
本申请描述的方法的步骤d)包括向稀释的乳清溶液中加入每升溶液约5克至10克、例如7克的热解硅石。
热解硅石为由团聚成具有特定化学物理性质的三级颗粒的非晶二氧化硅的微观液滴组成的特定类型的二氧化硅。通常以粉末形式存在于市场上的热解硅石为能够固定给定溶液中存在的脂蛋白、剩余脂肪物质和/或大分子的亲水热解硅石。这时,不需要详细描述热解硅石,因为其是本领域技术人员熟知且在各种技术领域广泛使用的。仅通过示例且不为限制本发明目的,热解硅石可为型,例如 380和/或 200。
加入以上二氧化硅之后,将每千克蛋白质约70至300ml乙醇(95%,体积/体积)加入溶液。例如,加入的乙醇的量为每千克蛋白质约120ml和甚至更多;例如,乙醇为食用级别。
之后,按照本申请描述的方法的步骤f),例如通过使用HCl将溶液的pH带至约4.5至5.0的值。步骤f)结束时溶液pH可为4.6、4.7、4.8、4.9、5.0的pH;例如pH将有4.6的值。
然后将这样获得的酸溶液加热至55℃与77℃之间的温度至少20分钟。特别地,该加热温度可为55℃、56℃、57℃、58℃、59℃、60℃、61℃、62℃、63℃、64℃、65℃、66℃、67℃、68℃、69℃、70℃,例如66℃。为了本发明的目的,本申请描述的加热(实际上包括将温度从乳清起始样本的初始温度升高到以上说明的相关温度)应在至少30分钟的间隔内发生。特别地,本方法的发明人观察到,更快的溶液加热决定了纯化的血清蛋白质的产率的损失,而更慢的溶液加热决定了血清蛋白质的不满意的纯化,因为不相关蛋白质(例如酪蛋白)没有被消除。
之后,将加热的溶液冷却到10℃与20℃之间、因此为10℃、11℃、12℃、13℃、14℃、15℃、16℃、17℃、18℃、19℃或20℃的更低温度。
将该冷却的溶液经历分离以分离包含血清蛋白质的部分。本申请描述的方法的该步骤i)可根据本领域中技术人员认为适于允许将蛋白质组分与以上溶液分离的任意一种分离/纯化技术来进行。仅通过示例,分离可通过使用Alfa laval分离器或Westfalia分离器连续进行。
然后将包含血清蛋白质的分离溶液的pH例如通过使用NaOH带至在5.8至6.8之间的值。该步骤l)结束时溶液pH会为5.8、5.9、6.0、6.1、6.2、6.3、6.4、6.5、6.6、6.7或6.8的pH;例如,pH会具有6.4的值。
然后,按照本说明书的目的的方法的步骤m),将血清蛋白质的溶液经历至少一个微孔过滤的过程,因此获得蛋白质浓度在100克/升与300克/升之间的蛋白质浓缩物。在本发明的一个实施例中,微孔过滤用截留为约12微米或0.6微米的膜进行。特别地,微孔过滤的至少一个第一过程可通过使用截留为12微米的膜来进行,接下来为使用截留为6微米的膜的微孔过滤的一个或多个过程。例如,本申请描述的方法中使用的微孔过滤膜为有机膜和/或陶瓷膜。
之后,将血清蛋白质的微孔过滤的溶液经历至少一个渗滤步骤,该渗滤步骤可与本说明书的方法的步骤b)中已经描述的步骤类似地进行。
最后,将微孔过滤和渗滤的乳清蛋白质溶液干燥和/或冷冻干燥,由此以乳清获得血清蛋白质浓缩物。
来自乳清的蛋白质的浓缩物或从上述方法可获得的血清蛋白质浓缩物的特别特征在于具有每千克蛋白质≤0.5克乳糖的浓度。此外,该方法产生了血清蛋白质浓缩物,其中血清蛋白质浓缩物中包含的蛋白质的纯度为至少96%。
此外,根据本申请公开的内容可获得的蛋白质浓缩物的特征还在于不具有乳清蛋白质典型具有的难闻气味和/或味道。
如上所示,该浓缩物的化学物理性质使其在需要使其中存在的乳糖的含量降低至最小值或例如不存在乳糖的条件(如其中其预期最终用途涉及对乳糖有不耐症和/或过敏症的受试者的情况)下特别有利。根据本申请已经提到的,本申请的目的还为包括上述蛋白质浓缩物的产品。特别地,该产品可用于任意领域,诸如,例如在不限制本申请的情况下,食品、化妆品、生物技术领域。仅通过示例,食品可为用于婴儿、生病受试者、儿童、老人或简单的为遵照特定节食方案的人的营养的食品,例如汤、酸奶、牛奶、果汁、点心等。在化妆品领域,反而,本发明的目的的浓缩物可包括在保湿产品中或通常在能给皮肤细胞提供营养的产品中。此外,在生物技术领域,血清蛋白质浓缩物可例如用作哺乳动物细胞或例如用于制备重组蛋白质的微生物的培养基中使用的元素。
该产品每千克蛋白质的浓度与本申请书的浓缩物的每千克蛋白质的浓度相同。
在根据本申请书的产品中,血清蛋白质为根据本申请的浓缩物中包括的血清蛋白质,且因此产品本身将具有等于每千克乳清蛋白质≤0.5克乳糖的乳糖浓度。
在包括本说明书的浓缩物的如牛奶或酸奶的产品的情况下,为了保持不存在乳糖和提高血清蛋白质的浓度,由此获得具有更大蛋白质浓度的产品,去乳糖牛奶可用作起始产品,将向该起始产品中加入本说明书的浓缩物。
示例
示例1:使食品富含乳清蛋白质浓缩物。
包括血清蛋白质浓缩物的食品可通过如下制备。
汤:对150ml与250ml之间的汤部分,10g与15g之间的浓缩物;
牛奶:对150ml与250ml之间的牛奶部分,5g与10g之间的血清蛋白质;
酸奶:对100ml与250ml之间的酸奶部分,5g与10g之间的浓缩物;
果汁:对100ml与1000ml之间的部分,5g与10g之间的浓缩物;
点心:对100ml与250ml之间的点心部分,5g与10g之间的浓缩物。
Claims (16)
1.一种以乳清样本制备具有降低的乳糖含量的可溶血清蛋白质浓缩物的方法,包括以下步骤:
a)浓缩所述乳清样本,因此获得在150克/升与300克/升之间的蛋白质浓度;
b)将所述乳清浓缩物经历至少一个渗滤步骤;
c)稀释渗滤的所述蛋白质浓缩物,因此获得蛋白质浓度在50克/升至90克/升范围内的溶液;
d)向所述溶液中加入每升溶液5克至10克的热解硅石;
e)将每千克蛋白质70ml至300ml的95%体积/体积乙醇加入所述溶液;
f)将步骤e)获得的所述溶液的pH酸化为4.5至5.0的值;
g)将酸化的所述溶液加热至55℃与70℃之间的温度至少20分钟,其中所述温度在30分钟内达到;
h)将所述溶液冷却至10℃与20℃之间的温度;
i)进行冷却的所述蛋白质溶液的分离;
l)使分离的所述溶液的pH至5.8至6.8的值;
m)将步骤l)获得的所述溶液经历至少一个微孔过滤的过程,因此获得蛋白质浓度在100克/升与300克/升之间的蛋白质浓缩物;
n)将所述蛋白质浓缩物经历至少一个渗滤步骤;
o)干燥所述蛋白质浓缩物,
其中来自乳清的所述蛋白质浓缩物的浓度为每千克蛋白质≤0.5克乳糖,并且
其中所述蛋白质的纯度为至少96%。
2.根据权利要求1所述的方法,其中所述乳清样本包括甜乳清和/或酸乳清。
3.根据前述权利要求1或2所述的方法,其中步骤a)和步骤n)的所述浓缩通过超滤来进行。
4.根据权利要求3所述的方法,其中所述超滤用截留为5000道尔顿与15000道尔顿之间的膜来进行。
5.根据权利要求1或2所述的方法,其中步骤b)和步骤n)中所述至少一个渗滤步骤为至少三个。
6.根据权利要求1或2所述的方法,其中步骤b)中所述至少一个渗滤步骤使用去矿物质水和/或去离子水和/或渗透溶液作为溶剂来进行。
7.根据权利要求6所述的方法,其中所述渗滤通过每体积浓缩蛋白质溶液使用3体积所述溶剂来进行。
8.根据权利要求1或2所述的方法,其中所述步骤d)通过每升所述稀释溶液加入7克热解硅石来进行。
9.根据权利要求1或2所述的方法,其中所述步骤e)通过将每千克蛋白质100ml至150ml乙醇加入所述溶液中来进行。
10.根据权利要求1或2所述的方法,其中所述步骤g)在66℃的温度下进行。
11.根据权利要求1或2所述的方法,其中步骤m)中的所述至少一个微孔过滤的过程使用截留为12微米或0.6微米的膜来进行。
12.根据权利要求1所述的方法,其中通过冷冻干燥的方式对所述蛋白质浓缩物进行干燥。
13.一种通过权利要求1至12中任一项所述的方法可获得的乳清蛋白质浓缩物。
14.根据权利要求13所述的乳清蛋白质浓缩物,每千克蛋白质包括≤0.5克乳糖。
15.根据权利要求13或14所述的乳清蛋白质浓缩物,其特征为不含有血清蛋白质典型具有的难闻气味和/或味道。
16.一种食品、化妆品或生物技术产品,包括根据权利要求13至15中任一项所述的乳清蛋白质浓缩物,其中所述产品具有每千克乳清蛋白质≤0.5克乳糖的浓度。
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IT000412A ITRM20120412A1 (it) | 2012-08-13 | 2012-08-13 | Metodo per la preparazione di un concentrato di proteine sieriche. |
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PCT/IB2013/056610 WO2014027305A1 (en) | 2012-08-13 | 2013-08-13 | A method for the preparation of a serum protein concentrate |
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US4229342A (en) * | 1977-05-18 | 1980-10-21 | Rhone-Poulenc Industries | Process for extracting proteins from milk using silica and anion exchange resins |
JPS61149053A (ja) * | 1984-12-24 | 1986-07-07 | Karupisu Shokuhin Kogyo Kk | 水親和性乳蛋白質 |
DK589785A (da) * | 1985-12-18 | 1987-06-19 | Samuelsson Ernst Gunnar | Peptidpraeparat, fremgangsmaade til fremstilling deraf samt anvendelse af peptidpraeparatet |
US5106836A (en) * | 1991-02-22 | 1992-04-21 | Clintec Nutrition Co. | Enteral diet |
JP3150504B2 (ja) * | 1993-02-26 | 2001-03-26 | 雪印乳業株式会社 | 低乳糖乳製品およびその製造方法 |
ZA949789B (en) * | 1993-12-30 | 1995-10-25 | Immunotec Res Corp Ltd | Process for making undenatured whey protein concentrate |
EP0933029A1 (en) | 1998-02-03 | 1999-08-04 | Xyrofin Oy | Skimmed milk powder substitute |
NZ501676A (en) * | 1999-12-09 | 2002-12-20 | New Zealand Dairy Board | Calcium-depleted milk protein products and use in cheese manufacture to reduce nugget-formation |
US20030026845A1 (en) * | 2001-06-18 | 2003-02-06 | Etzel Lisa R. | Process for preparing protein isolate from milk, whey, colostrum, and the like |
US20050196508A1 (en) * | 2004-03-05 | 2005-09-08 | Joseph Wang | Lactose-removed milk product and process for the preparation thereof |
CN101019580B (zh) * | 2007-03-12 | 2010-07-14 | 苏州科牧动物药品有限公司 | 一种高含量免疫球蛋白乳清粉的制备方法 |
CN101491287B (zh) * | 2008-01-24 | 2012-05-23 | 滕国新 | 从乳清中提取乳糖和乳清蛋白的方法 |
US9055752B2 (en) * | 2008-11-06 | 2015-06-16 | Intercontinental Great Brands Llc | Shelf-stable concentrated dairy liquids and methods of forming thereof |
US20130011515A1 (en) | 2009-10-19 | 2013-01-10 | Cytosport, Inc. | Milk protein concentrates |
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BR112015003229A2 (pt) | 2017-08-08 |
ES2588627T3 (es) | 2016-11-03 |
EP2882305A1 (en) | 2015-06-17 |
JP6258323B2 (ja) | 2018-01-10 |
EA031775B1 (ru) | 2019-02-28 |
ITRM20120412A1 (it) | 2014-02-14 |
CA2881782C (en) | 2022-02-15 |
JP2015524668A (ja) | 2015-08-27 |
CN104582508A (zh) | 2015-04-29 |
US9534028B2 (en) | 2017-01-03 |
HUE030043T2 (en) | 2017-04-28 |
EA201590275A1 (ru) | 2015-05-29 |
AU2013303829A9 (en) | 2015-05-07 |
US20150203552A1 (en) | 2015-07-23 |
WO2014027305A1 (en) | 2014-02-20 |
BR112015003229B1 (pt) | 2020-12-01 |
NZ704744A (en) | 2017-11-24 |
WO2014027305A9 (en) | 2015-04-09 |
EP2882305B1 (en) | 2016-07-06 |
AU2013303829A1 (en) | 2015-02-26 |
AU2013303829B2 (en) | 2015-09-03 |
CA2881782A1 (en) | 2014-02-20 |
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