CN1625566B - 乳铁传递蛋白 - Google Patents
乳铁传递蛋白 Download PDFInfo
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- CN1625566B CN1625566B CN028288009A CN02828800A CN1625566B CN 1625566 B CN1625566 B CN 1625566B CN 028288009 A CN028288009 A CN 028288009A CN 02828800 A CN02828800 A CN 02828800A CN 1625566 B CN1625566 B CN 1625566B
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- Prior art keywords
- lactoferrin
- polypeptide
- bone
- milk
- per molecule
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Abstract
本发明公开了纯的每分子含有不多于2个金属离子的乳铁传递蛋白多肽或者含有该多肽及其片段的混合物。该多肽或该混合物能刺激骨骼生长和抑制骨吸收。同时本发明还公开了利用该多肽或该混合物治疗与骨骼相关的病症的方法。
Description
相关申请
本申请要求申请号为518121、申请日为2002年4月3日的新西兰申请的优先权，本文引用其内容作为参考。
背景技术
乳铁传递蛋白是一种80KD的铁结合糖蛋白，存在于包括眼泪、胆汁、支气管粘液、胃肠液、子宫颈-阴道粘液、精液及乳汁在内的大多数外分泌液中。它是循环中多型核中性粒细胞次级特异颗粒的主要成分。乳铁传递蛋白最丰富的来源是哺乳动物的乳汁和初乳。
乳铁传递蛋白在循环中的含量为2-7μg/ml。它可能具有多种生物学功能，包括铁代谢、免疫功能及胚胎发育的调节等。乳铁传递蛋白对包括革兰氏阳性和革兰氏阴性细菌、酵母及真菌在内的多种病原体具有抗微生物活性。乳铁传递蛋白的抗微生物作用源于其与病原体生长必需的铁的结合能力。乳铁传递蛋白还能抑制多种病毒的复制并可通过与细菌膜上脂多糖的脂质体A成分结合来增加某些细菌对抗生素的敏感性。
发明概述
本发明涉及具有刺激骨骼生长和抑制骨吸收作用的乳铁传递蛋白多肽。特别地，本发明涉及每分子含有不多于2(即0、1或优选2)个金属离子的纯的乳铁传递蛋白多肽。“纯的”多肽指不含有其它生物大分子的多肽并且其在干重时的含量至少为65％(例如，至少为70％、75％、80％、85％、90％、95％或99％)纯度。可通过任何适合的标准方法来检测多肽的纯度，例如通过柱层析、聚丙烯酰氨凝胶电泳或HPLC分析。此乳铁传递蛋白多肽可以是自然产生的多肽、重组多肽或合成的多肽。保持野生型乳铁传递蛋白多肽生物学活性的野生型乳铁传递蛋白多肽的变体(例如含有至少2(例如4、6、8、10、20、50、100、200、300、400、500、 600、700)个氨基酸的野生型乳铁传递蛋白多肽片段或含有乳铁传递蛋白多肽序列的重组蛋白)也在本发明的范围内。本发明中的乳铁传递蛋白多肽可以是哺乳动物来源的，例如来自人或牛的乳汁。与多肽结合的金属离子可以是铁离子(如自然产生的乳铁传递蛋白多肽中)、铜离子、铬离子、钴离子、锰离子、锌离子或镁离子。
本发明中的乳铁传递蛋白多肽可用于刺激骨骼生长(例如，通过促进成骨细胞和软骨细胞的增殖)和抑制骨吸收(例如，通过抑制破骨细胞的生长)。本发明制备的乳铁传递蛋白多肽(例如，从牛奶中分离的乳铁传递蛋白)可包括单一种类的多肽，例如每分子结合2个铁离子的多肽。其还可包含不同种类的多肽，例如其中一些分子不结合离子而其它分子每分子结合1个或2个离子；一些分子结合铁离子而其它分子结合铜离子；一些分子是含0、1或2个金属离子的具有生物活性的乳铁传递蛋白多肽(全长或短于全长)而其它分子为该多肽的片段(相同或不同)；或所有分子为含0、1或2个金属离子的全长乳铁传递蛋白多肽的片段(相同或不同)。例如，全长乳铁传递蛋白多肽和全长乳铁传递蛋白多肽的各种片段的混合物可从全长乳铁传递蛋白多肽的水解产物，例如诸如蛋白酶消化的不完全消化产物中制备。或者，该混合物可通过将全长乳铁传递蛋白多肽与其各种片段(例如，合成片段)混合来制备。另一方面，全长乳铁传递蛋白多肽的各种片段的混合物，也可通过例如全长乳铁传递蛋白多肽的完全消化(即消化后没有全长多肽存在)或全长乳铁传递蛋白多肽的不同片段的混合来制备。
本发明还涉及一种营养(nutraceutical)组合物，其可以是乳汁、果汁、饮料、快餐或食品补充剂(dietary supplement)。该营养组合物含有高于自然含量的本发明乳铁传递蛋白多肽或该多肽与其片段的混合物。已发现乳铁传递蛋白可刺激成骨细胞和软骨细胞的增殖并抑制破骨细胞的生长。因此，本发明中的营养组合物可用于预防和治疗诸如骨质疏松、类风湿或骨性关节炎的骨性病症。该营养组合物还可包括适当含量的另一种诸如钙、锌、镁维生素C、维生素D、维生素E、维生素K2或其混合物的骨增强成分。
此外，本发明还涉及一种药物组合物，含有本发明的乳铁传递蛋白 多肽或该多肽与其片段的混合物以及药物可接受的载体。可选择地，该药物组合物还可包含其它骨增强剂。本发明还涉及上述乳铁传递蛋白多肽或该多肽与其片段的混合物在生产预防和治疗骨性疾病的药物方面的用途。
本发明提供了预防和治疗骨相关病症的方法(例如通过刺激骨骼生长和抑制骨吸收)。该方法包括给予个体其所需有效剂量的本发明的乳铁传递蛋白多肽或该多肽与其片段的混合物。该方法还包括同时给予该个体有效剂量的另一种骨增强剂。
本发明中一个或多个实施方案的详细描述如下所述。本发明的其它特征、目的和优点可从详细描述和权利要求中得出。
详细描述
本发明是基于意外发现的乳铁传递蛋白刺激成骨细胞和软骨细胞增殖并抑制破骨细胞生长的特性。因此，乳铁传递蛋白可用于预防和治疗骨性病症。
本发明的乳铁传递蛋白多肽是纯的每分子含有不多于2个金属离子的多肽。事实上，制备的乳铁传递蛋白的离子/乳铁传递蛋白比值的检测表明其比值可在0-2.5范围。其可从天然来源(例如哺乳动物乳汁)中分离得到或利用本领域所属技术人员公知的遗传工程或化学合成技术生产。以下就是从牛奶中分离乳铁传递蛋白的示范步骤：
4℃下以5ml/min流量，使新鲜的脱脂乳汁(7L，pH6.5)通过用超纯水(milli Q water)平衡的300ml的S Sepharose Fast Flow柱。用2.5倍床体积的水冲洗除去未结合蛋白，并用约2.5倍床体积的分别为0.1M、0.35M和1.0M的NaCl将结合蛋白逐步洗脱。收集1M NaCl洗脱所得的粉红乳铁传递蛋白条带作为单一组分，并用milli Q水透析随后冻干。将该冻干粉溶解在25mM、pH6.5磷酸钠缓冲液中，并在S Sepharose FastFlow柱中，以3ml/min流量、使用NaCl浓度梯度到1M的上述缓冲液再层析。经过凝胶电泳和反向HPLC分析，将含足够纯度的乳铁传递蛋白的组分合并在一起，透析并冻干。在pH8.6、含0.15M KCl的80mM磷酸二钾溶液中，通过在Sephacryl 300上进行凝胶过滤得到最终的乳铁传 递蛋白纯品。合并所选择的组分，用milli Q水透析并冻干。HPLC分析结果和乳铁传递蛋白的铁离子饱和形式的光谱比值(280nm/465nm)为～19或更低的事实表明该制备所得的乳铁传递蛋白纯度大于95％。
通过在pH7.8含1％纯化乳铁传递蛋白的50mM Tris、10mM碳酸氢钠溶液中以摩尔比1∶2加入过量的5mM次氮基三乙酸铁(ferricnitrilotriacetate)可实现乳铁传递蛋白的铁离子饱和(Foley and Bates(1987)Analytical Biochemistry 162，296-300)。在4℃下，用100倍体积的milli Q水(更新两次)透析20小时，以除去过量的次氮基三乙酸铁。然后将该铁离子负载的(holo-)乳铁传递蛋白冻干。
在4℃下，使用30倍体积的pH2.3含0.1M柠檬酸和500mg/L EDTA二钠的溶液，对1％的高度纯化乳铁传递蛋白样品的水溶液透析30小时，从而制备去除铁离子的(apo-)乳铁传递蛋白(Massons and Heremans(1966)Protides of the Biological fluids 14，115-124)。用30倍体积的milli Q水(更新一次)透析除去柠檬酸盐和EDTA，并将得到的无色溶液冻干。
本发明中的乳铁传递蛋白多肽可以含有铁离子(如在自然产生的乳铁传递蛋白多肽中)或非铁金属离子(如铜离子、铬离子、钴离子、锰离子、锌离子或镁离子)。例如，可从由牛奶中分离的乳铁传递蛋白中除去铁离子，然后加载其它类型的金属离子。例如，可通过与上述加载铁离子相同的方法实现铜离子的加载。可使用Ainscough等人的方法((1979)Inorganica Chimica Acta33，149-153)在乳铁传递蛋白上加载其它金属离子。
在本发明中乳铁传递蛋白多肽的制备中，所述多肽可以是单一种类或不同种类。例如，该多肽中每个都可含有不同数目的金属离子或不同种类的金属离子；或该多肽的长度可以不同，例如，一些是全长多肽而另一些是片段，且该片段中每个都能代表全长多肽的特定部分。可以从自然来源或通过不同种类乳铁传递蛋白多肽的混合来获得此种制剂。例如，通过对全长乳铁传递蛋白多肽的蛋白酶消化(完全或部分)来制备不同长度乳铁传递蛋白多肽的混合物。可根据本领域所属技术人员公知的方法(如控制蛋白酶的用量或孵育时间)控制消化的程度。完全消化能产生全长乳铁传递蛋白多肽的各种片段的混合物；部分消化能产生全长乳铁传 递蛋白多肽与其各种片段的混合物。
如上所述的乳铁传递蛋白多肽或该多肽与其片段的混合物用于制备本发明中预防和治疗骨相关病症的营养组合物。该病症的例子包括但不限于骨质疏松、类风湿或骨性关节炎、肝性骨营养不良、骨质软化症、佝偻病、囊性纤维性骨炎、肾性骨营养不良、骨硬化、骨质减少、骨成纤维发生不全(fibrogenesis-imperfecta ossium)、次级甲状旁腺功能亢进、甲状旁腺功能减退、甲状旁腺功能亢进、慢性肾病、结节病(sarcoidosis)、糖皮质激素诱导的骨质疏松症、特发性高钙血症、派杰病(Paget’s disease)及成骨不全。该营养组合物可以是食品补充剂(如胶囊、小袋或片剂)或食品(如乳汁、果汁、饮料、草药茶叶袋或糖果)。该组合物还可包括诸如蛋白、碳水化合物、维生素、微量元素或氨基酸的其它营养成分。该组合物可以是适宜口服的诸如片剂、硬或软胶囊、水或油悬浮液或糖浆的形式，或者是适宜肠道外使用的诸如丙二醇水溶液、或缓冲水溶液的形式。营养组合物中活性成分含量的变化很大程度上取决个体的特定需求。如本领域所属技术人员所公认的，根据不同的给药途径及可能的与其它骨增强剂的共同使用，所述活性成分的含量也可不同。
本发明还包括一种药物组合物，含有上述的乳铁传递蛋白多肽或该多肽与其片段的混合物以及药物可接受的载体。该药物组合物可用于预防和治疗上述的骨相关病症。该药物组合物还可包括有效剂量的其它骨增强剂。所述药物可接受的载体包括溶剂、分散介质、包衣、抗菌和抗真菌剂、等张和吸收延迟剂。有效剂量是产生治疗效果所需的剂量。动物剂量和人类剂量的相互关系(根据每平方米体表面积的毫克数)如Freireich等人(1966)在Cancer Chemother.Rep.50：219中所述。可根据个体的身高和体重来估算其体表面积，参见例如Scientific Tables，GeigyPharmaceuticals，Ardley，New York，1970，537。如本领域所属技术人员所公认的，根据不同的给药途径、赋形剂和其类似物的使用，所述有效剂量也可不同。
本发明中的乳铁传递蛋白多肽或该多肽与其片段的混合物可通过常规方法组方到用于不同给药途径的剂型中。例如，它可制成口服给药的胶囊、胶封或片剂。胶囊可含有任何常规的诸如凝胶或纤维素的药物可 接受的物质。片剂可通过常规的步骤将乳铁传递蛋白多肽混合物或该多肽与其片段的混合物与固体载体和润滑剂的混合物压片制成。固体载体的例子包括淀粉和糖膨润土(bentonite)。本发明的乳铁传递蛋白多肽或该多肽与其片段的混合物还能以含有结合剂的硬壳片剂或胶囊的形式给药，该结合剂如乳糖或甘露醇、常规的填充剂及片剂形成剂。本发明的药物组合物可通过肠道外给药。肠道外给药剂型的例子包括水溶液、含活性成分的等张盐水或5％葡萄糖、或其它公知的药物可接受赋形剂。环糊精或其它本领域所属技术人员公知的增溶剂可用做释放该治疗性成分的药物赋形剂。
本发明组合物的有效性可在体外和体内评价。如见以下的实施例。简而言之，能检测到该组合物体外促进成骨细胞和软骨细胞增殖的能力。对于体内的研究，可将该制剂注入动物(例如小鼠)体内并随后评价其对骨组织的作用。根据结果可确定适当的剂量范围和给药途径。
以下的特定实施例仅是为说明而进行的解释，并不对本发明其它部分的构成任何限制。不需要进一步的创造性劳动，本领域所属技术人员就可根据本说明书，使用本发明到极限。本文所引用的出版物均在此全部引用作为参考。
乳铁传递蛋白促进原代大鼠成骨细胞增殖的作用
根据Lowe与其同事所述的方法(Lowe，et al.(1991)Journal of Boneand Mineral Research 6，1277-1283)，从20天的大鼠乳鼠的颅盖骨中用胶原酶消化分离成骨细胞。无菌切下颅盖骨，并剥离额骨和顶骨的骨膜。只收集没有骨缝组织的中间部分。该颅盖骨在37℃摇动的水浴中用含3mM EDTA(pH7.4)的磷酸盐缓冲液(PBS)处理2次，15分钟。用PBS冲洗一次之后，37℃下将该颅盖骨用3ml 1mg/ml胶原酶处理两次，7分钟。在丢弃第一和第二次消化的悬浮液后，该颅盖骨进一步用3ml 2mg/ml胶原酶处理2次(30分钟、37℃)。收集第三和第四次消化的悬浮液、离心并用含10％胎牛血清(FCS或FBS)的Dulbecco′smodified Eagle′培养液(DME)洗涤细胞，然后悬浮在DME/10％FCS培养液中放入75cm3培养瓶中。将该细胞在5％CO2、95％空气、37℃下进行培养。5-6天后可形成融合单层，此时进行传代培养。用胰蛋白酶-EDTA(0.05％/0.53mM)进行胰蛋白酶消化后，在含5％FCS的minimum essential培养液(MEM)中漂洗细胞，并重悬在新鲜的培养液中，然后以5×104细胞/ml密度接种于24孔板中(每孔0.5ml细胞悬浮液，即1.4×104细胞/cm2)。碱性磷酸酶活性和骨钙素产生的高水平[见Groot，et al.(1985)Cell Biol Int Res 9，528]及腺苷酸环化酶对甲状旁腺激素与前列腺素具有敏感性反应[见Hermann-Erlee，etal.(1986)Ninth International Conference on calcium regulating hormonesand bone metabolism，p409]的鉴定结果，表明这些细胞具有成骨细胞样特性。
在活跃生长细胞群和非生长活跃细胞群中进行了增殖研究(细胞记数和胸腺嘧啶脱氧核苷掺入)。为产生活跃生长细胞，将亚融合单层的细胞群(传代培养后24h)放入新鲜的含1％FCS和一种乳铁传递蛋白样品的MEM培养液中。为产生非活跃生长的细胞，将亚融合单层的细胞群放入新鲜的含0.1％牛血清白蛋白(BSA)和一种乳铁传递蛋白样品的无血清培养液中。在加入如上述所制备的乳铁传递蛋白样品(即纯化的乳铁传递蛋白、holo-乳铁传递蛋白及apo-乳铁传递蛋白)后6、24和48小时进行细胞记数分析。在37℃，通过将细胞暴露于胰蛋白酶/EDTA(0.05％/0.53mM)约5分钟将细胞从培养板的孔中分离，然后进行细胞记数。在血球记数小室中进行记数。在培养终止前2小时，将[3H]-胸腺嘧啶脱氧核苷(1μCi/孔)与细胞孵育，进行[3H]-胸腺嘧啶脱氧核苷对生长活跃细胞和非生长活跃细胞的掺入实验。用含冷的胸腺嘧啶脱氧核苷的MEM洗涤细胞并随后加入10％三氯乙酸在6、24或48小时终止实验。用乙醇∶乙醚(3∶1)洗涤沉淀2次，并在室温下干燥小孔。在55℃，用2M KOH重溶解残余物30分钟，用1M HCl中和，然后取一份用于放射性记数。对于细胞记数和胸腺嘧啶脱氧核苷掺入，每个实验在每个时间点都对至少含6个小孔的实验组进行4次不同的检测。
成骨细胞增殖率的显著增加(即掺入到生长细胞DNA中的胸腺嘧啶脱氧核苷增加)表明纯化乳铁传递蛋白样品对细胞的有丝分裂反应非常有效。将上述观察到的有效的成骨反应与胰岛素样生长因子1(IGF-1，已知的成骨细胞有丝分裂素)的成骨反应进行对比。在相同成骨细胞培养系统
条件下，IGF-1显示的最大效果为对照组的1.25倍，而乳铁传递蛋白在最高实验剂量(10μg/ml)的效果是对照组的2.26倍。
乳铁传递蛋白促进软骨细胞增殖的作用
在无菌条件下，通过移去绵羊胫骨和股骨表面的软骨(全厚度片)分离出软骨细胞。骨片放置在含5％FBS(v/v)和抗生素(50g/L青霉素、50g/L链霉素及100g/L新霉素)的DME培养液中并用手术刀片小心切碎。移去组织并在37℃先用链霉蛋白酶(0.8％w/v、90分钟)孵育，随后用胶原酶(0.1％w/v、18小时)孵育完成消化。通过离心(10分钟、1300rpm)从消化液中分离细胞，并重悬在DME/5％FBS中，用90Fm孔径的尼龙网过滤除去未消化的碎片，并再次离心。随后将细胞用相同的培养液洗涤和重悬2次，并接种于含DME/10％FBS培养液的75cm3培养瓶中，在5％CO2、95％空气、及37℃条件下培养。7天后可形成融合单层，此时进行传代培养。用胰蛋白酶-EDTA(0.05％/0.53mM)进行胰蛋白酶消化后，在DME/5％FBS培养液中漂洗细胞，并重悬在新鲜的培养液中，然后接种于24孔板中(5×104细胞/mL，0.5ml/孔)。对生长停止的细胞群进行的胸腺嘧啶脱氧核苷掺入检测如上述的成骨细胞样细胞培养所述。乳铁传递蛋白在高于0.1μg/ml浓度时可刺激软骨细胞的增殖。
乳铁传递蛋白在器官培养中促进成骨细胞的增殖
新生小鼠的器官培养见以往报道(Comish，et al.(1998)Am J Physiol274，E827-E833)。简而言之，2天大的新生小鼠皮下注入放射性标记的 45Ca。3天后，切下颅盖骨并放在含0.1％牛血清白蛋白/Media199的Petri培养皿中的网格上。加入乳铁传递蛋白，并培养颅盖骨48小时。在培养时间结束前4小时，加入[3H]-胸腺嘧啶脱氧核苷。终止实验，并分析45Ca释放和胸腺嘧啶脱氧核苷掺入量。发现乳铁传递蛋白可刺激反映成骨细胞系细胞增殖的DNA合成作用。
通过成骨细胞中MAP激酶的乳铁传递蛋白信号
该方法以往已有报道(Grey，et al.(2001)Endocrinology 142，1098-1106)。特别是，如上所述制备的原代大鼠成骨细胞以5×104细胞/ml初始密度接种于6孔组织培养板中，并在5％FCS的MEM培养液中生长成为80-90％的融合单层。在血清饥饿过夜后，在室温用含乳铁传递蛋白的MEM/0.1％BSA处理细胞。在乳铁传递蛋白诱导的p42/44 MAP激酶磷酸化的信号转导抑制剂的作用的鉴定实验中，加入乳铁传递蛋白前，将细胞用抑制剂预处理30分钟。在完成所指示时间的处理后，吸除培养液，用冰冷的PBS洗涤细胞，随后用冰冷的含蛋白酶抑制剂与磷酸酶抑制剂的组合(1mM PMSF、1μg/ml peptatin、10μg/ml leupeptin，10μg/ml抑肽酶、1mM钒酸钠、500mM NaF)的HNTG裂解缓冲液(50mM HEPES、pH7.5，150mM NaCl，1％Triton，10％甘油，1.5mM MgCl2，1mM EDTA)刮下细胞。裂解液经短暂的涡旋处理，以13,000rpm、4℃离心，随后储存于-70℃直到检测。通过DC蛋白分析(BioRad，Hercules，CA)来检测细胞裂解液中的蛋白含量。用相同量的全细胞裂解液(30-50μg)进行8％SDS-PAGE电泳，并转移至硝化纤维素膜，用抗磷酸化-p42/44 MAP激酶的抗体(1∶1000)在4℃免疫标记过夜。作为对照蛋白，取相同的过滤液(filters)并用抗全部p42/44MAP激酶的抗体(1∶400)再标记。将所述硝化纤维素膜用HRP-连接二抗在室温下孵育1h，并用ECL分析。免疫标记重复至少3次。发现在1-100μg/ml浓度范围内，乳铁传递蛋白对成骨细胞中p42/44 MAP激酶的磷酸化呈现出与剂量和时间相关的诱导作用。
乳铁传递蛋白在体内刺激骨骼生长
这些研究中使用的小鼠模型如以往的报道所述(Comish，et al.(1993)Endocrinology 132，1359-1366)。每天注入乳铁传递蛋白(0mg、0.04mg、0.4mg及4mg)，共5天，并在一周后将动物处死。通过新形成骨骼的荧光标记来确定骨骼的形成。通过常规的光学显微镜来确定骨吸收和骨聚集指数，并辅以软件图像分析。对成年小鼠的乳铁传递蛋白的局部注入可增加颅盖骨的生长，并仅在5次注入后就可显著增加骨的面积。
应用例1
含14％到17％固体的酸乳酪块(加入或不加入水果)可用如下方法制 备：
中等热度的脱脂奶粉(109-152g)和ALACO稳定剂(100g)复溶于约880ml、50℃的水中。随后加入无水的乳脂(20g)并混合30分钟。然后将该混合物加热至60℃，在200bar压力下研磨，并在90℃进行巴氏消毒。冷却到40-42℃温度后，加入如上所述的起始混合物和冻干蛋白制剂(95％纯度的最多到50mg的乳铁传递蛋白或相等量的未达到如此高纯度的原料)。如果需要，此时可加入新鲜的水果。随后将该混合物装入容器，并在40℃孵育直到pH4.2-4，并在吹风器中冷却。
可选择的制备该相同酸乳酪块的方法是在其用于制备酸乳酪之前，将干燥的指定数量或剂量比值的乳铁传递蛋白搅拌到干燥的奶固体中。
应用例2
脱脂或全脂奶粉与钙质及上述冻干乳铁传递蛋白制剂的干混合物可制成奶制品制剂或组合物，其可用做功能食品或功能食品成分。此种组合物可用于再生奶、奶粉成分、奶制甜点、功能食品、奶酪或黄油或饮料、和营养制剂或食品补充剂。奶粉∶钙质∶活性乳铁传递蛋白成分的干重混合比例在90∶9.5∶0.5与94∶5.95∶0.0001之间的混合物，可提供适宜此种用途的组合物。
应用例3
由奶粉、钙质和丰富的乳铁传递蛋白成分混合的组合物可用做骨骼保健功能食品、骨骼保健食品成分或作为一些保健食品中释放骨骼保健营养素的食品成分。
对于这类组合物，其钙含量和蛋白含量应调整到所需的和允许的营养限度。商业销售的成分奶粉，根据它们的来源，通常每100g奶粉中含有300和900mg钙。可在奶粉中加入钙源使钙含量增加到成分奶粉混合物重量的3％。商业销售的成分奶粉或奶制品蛋白粉中的蛋白含量根据该成分的类型、制造方法和使用需求的不同而变化。成分奶粉通常含有12％～92％的蛋白。例子包括商业销售的脱脂和全脂奶粉、食品级酪蛋白、金属酪蛋白、乳汁蛋白浓缩粉、喷雾干燥超滤或微过滤滞留粉及乳汁蛋 白分离产物。所述富含乳铁传递蛋白制剂可加入到蛋白和钙质的混合物中来制备营养奶粉，该奶粉可用做保健食品和饮料的成分。此种混合物适宜作为制备酸乳酪和酸乳酪饮料、酸性饮料、成分奶粉混合物、巴氏消毒液体奶制品、UHT奶制品、培养奶制品、酸奶饮料、奶和谷类组合产品、麦芽奶、豆奶组合产品的成分使用。对于这些用途，混合物中的钙含量可为0.001％～3.5％(w/w)，蛋白含量为2％～92％，作为成骨细胞增殖成分的乳铁传递蛋白以0.000001％～5.5％的含量加入。
其它实施方案
本说明书公开的所有特征能以任何组合方式进行组合。本说明书公开的每个特征可被实现相同、相等或相似目的的可选择的特征代替。因此，除非特别说明，否则所公开的每个特征只是系列相等或相似特征的实施例。
如上所述，本领域所属技术人员可显而易见地确定本发明的基本特征，并在不偏离本发明精神和范围的情况下，对本发明做出各种改变和修改以适应各种用途和条件。因此，其它实施方案同样属于本发明权利要求的保护范围。
Claims (6)
1.每分子含有不多于2个铁离子的人乳铁传递蛋白或牛乳铁传递蛋白在制备刺激骨骼生长和抑制骨吸收的营养或药物组合物中的用途，其中所述的乳铁传递蛋白不含有其他生物大分子，为干重时至少65％的纯度。
2.如权利要求1所述的用途，其中所述乳铁传递蛋白每分子含有1个铁离子或每分子含2个铁离子。
3.如权利要求1或2所述的用途，其中所述组合物为口服剂型或肠道外剂型。
4.每分子含有不多于2个铁离子的人乳铁传递蛋白或牛乳铁传递蛋白在制备治疗骨相关病症的营养或药物组合物中的用途，其中所述乳铁传递蛋白不含有其他生物大分子，为干重时至少65％的纯度，其中所述骨相关病症为骨质疏松或骨性关节炎、骨质软化症、骨质减少、骨成纤维发生不全、糖皮质激素诱导的骨质疏松症。
5.如权利要求4所述的用途，其中在所述乳铁传递蛋白每分子含有1个铁离子或每分子含2个铁离子。
6.如权利要求4或5所述的用途，其中所述组合物为口服剂型或肠道外剂型。
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| NZ51812102 | 2002-04-03 | ||
| PCT/NZ2002/000137 WO2003082921A1 (en) | 2002-04-03 | 2002-07-29 | Lactoferrin |
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| US20030191193A1 (en) * | 2002-04-03 | 2003-10-09 | Jillian Cornish | Lactoferrin |
| NZ536338A (en) * | 2002-11-29 | 2006-04-28 | Morinaga Milk Industry Co Ltd | Lactoferrin as a cysteine protease inhibitor |
| US7956031B2 (en) | 2005-05-31 | 2011-06-07 | Naidu Lp | Metallo-lactoferrin-coenzyme compositions for trigger and release of bioenergy |
| TWI276442B (en) * | 2005-07-05 | 2007-03-21 | Maxluck Biotechnology Corp | Composition of controlling and preventing heart disease |
| EP1937298A4 (en) * | 2005-10-14 | 2009-11-11 | Auckland Uniservices Ltd | USE OF LACTOFERRIN FRAGMENTS AND HYDROLYSATES |
| US20090130191A1 (en) * | 2005-10-27 | 2009-05-21 | Sunstar Inc. | Osteoclast Growth Inhibitor, Oral Composition, and Preventive or Therapeutic Agent for Bone Diseases, Containing Liposome-Encapsulated Lactoferrin |
| US8021659B2 (en) | 2006-04-28 | 2011-09-20 | Naidu Lp | Coenzyme Q10, lactoferrin and angiogenin compositions and uses thereof |
| FR2903906B1 (fr) * | 2006-07-19 | 2010-11-05 | Bio Serae Laboratoires | Composition pour prevenir et/ou traiter les maladies articulaires degeneratives. |
| WO2008094493A2 (en) * | 2007-01-26 | 2008-08-07 | Ventria Bioscience | Compositions containing lactoferrin and calcium, and methods of using same |
| JP2008303168A (ja) * | 2007-06-07 | 2008-12-18 | Japan Health Science Foundation | 破骨細胞制御剤とそのスクリーニング方法 |
| ES2518290T3 (es) | 2007-07-10 | 2014-11-04 | Glanbia Nutritionals (Ireland) Limited | Método de eliminación de endotoxinas de proteínas |
| US8614189B2 (en) * | 2008-09-24 | 2013-12-24 | University Of Connecticut | Carbon nanotube composite scaffolds for bone tissue engineering |
| MX2011005527A (es) | 2008-11-28 | 2011-09-28 | Univ Otago | Uso de la bacteria de acido lactico para tratar o prevenir el eczema. |
| FR2950510B1 (fr) * | 2009-09-28 | 2013-04-12 | Groupe Lactalis | Derive laitier pour son utilisation dans le developpement et/ou le maintien de la condition physique des mammiferes, en particulier des animaux de competition. |
| US8545927B2 (en) | 2010-05-10 | 2013-10-01 | University Of Connecticut | Lactoferrin-based biomaterials for tissue regeneration and drug delivery |
| WO2015000694A1 (en) * | 2013-07-05 | 2015-01-08 | Nestec S.A. | Lactoferrin-osteopontin-iron complex |
| US10018418B2 (en) | 2014-01-07 | 2018-07-10 | Can-Eng Partners Limited | Mobile removable hearth for furnace and transporter |
| EP4136985A1 (en) | 2016-12-22 | 2023-02-22 | University of Otago | Use of lactic acid bacteria to treat or prevent gestational diabetes mellitus |
| PH12019501439B1 (en) | 2016-12-22 | 2024-02-23 | Univ Of Otago | Use of lactic acid bacteria to treat or prevent gestational diabetes mellitus |
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- 2002-07-29 WO PCT/NZ2002/000137 patent/WO2003082921A1/en active Application Filing
- 2002-07-29 BR BR0215682-2A patent/BR0215682A/pt not_active IP Right Cessation
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Also Published As
| Publication number | Publication date |
|---|---|
| EP1490404A4 (en) | 2010-02-24 |
| US20030191193A1 (en) | 2003-10-09 |
| CN1625566A (zh) | 2005-06-08 |
| US8703699B2 (en) | 2014-04-22 |
| CA2481315A1 (en) | 2003-10-09 |
| AR040695A1 (es) | 2005-04-13 |
| HK1074635A1 (en) | 2005-11-18 |
| US20090259025A1 (en) | 2009-10-15 |
| JP4327607B2 (ja) | 2009-09-09 |
| PT1490404E (pt) | 2013-09-24 |
| EP1490404B1 (en) | 2013-08-21 |
| WO2003082921A1 (en) | 2003-10-09 |
| ES2435071T3 (es) | 2013-12-18 |
| US20110183008A1 (en) | 2011-07-28 |
| EP1490404A1 (en) | 2004-12-29 |
| JP2006501143A (ja) | 2006-01-12 |
| US20080188644A1 (en) | 2008-08-07 |
| BR0215682A (pt) | 2005-01-04 |
| AU2002324387A1 (en) | 2003-10-13 |
| DK1490404T3 (da) | 2013-09-16 |
| US20100305308A1 (en) | 2010-12-02 |
| KR20050006140A (ko) | 2005-01-15 |
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