CN101842026A - 骨强化用食品原材料 - Google Patents
骨强化用食品原材料 Download PDFInfo
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- CN101842026A CN101842026A CN200880113764A CN200880113764A CN101842026A CN 101842026 A CN101842026 A CN 101842026A CN 200880113764 A CN200880113764 A CN 200880113764A CN 200880113764 A CN200880113764 A CN 200880113764A CN 101842026 A CN101842026 A CN 101842026A
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- fraction
- bone
- lactoprotein
- lactoprotein fraction
- amino acid
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Abstract
本发明涉及一种骨强化剂,其由具有以下(1)~(4)特性的乳蛋白质级分构成。(1)其为乳来源。(2)其为含有分子量在6,000~150,000道尔顿范围内的蛋白质成分的级分。(3)构成氨基酸的组成中含有12~14重量%的碱性氨基酸、且碱性氨基酸/酸性氨基酸的比在0.5~0.7的范围内。(4)具有成骨细胞的钙化促进作用。
Description
技术领域
本发明涉及具有骨强化作用的乳蛋白质级分或乳蛋白质级分分解物。
本发明的乳蛋白质级分或乳蛋白质级分分解物具有骨强化作用,因而作为以骨疾病的预防或治疗、骨强化等为目的的骨强化剂,或者作为骨疾病的预防或治疗用或骨强化用药品、饮食品和饲料的有效成分非常有用。
背景技术
近年来,伴随着高龄化,骨质疏松症、骨折、腰痛等各种骨疾病具有增加的倾向。在骨组织中,不断进行着骨生成和骨吸收,在年轻时骨生成和骨吸收保持着良好的平衡,但随着年龄的增长,由于各种原因该平衡倾向于骨吸收(解偶联)。并且,长时间持续该状态的话会使得骨组织变脆,导致发生骨质疏松症、骨折、腰痛等各种骨疾病。认为如果能够防止该解偶联,则能够预防骨质疏松症、骨折、腰痛等各种骨疾病。
以往,作为防止该解偶联、预防或治疗各种骨疾病的方法,曾进行了以下方法:(1)食物补钙,(2)轻微运动,(3)日光浴,(4)药物治疗等。对于食物补钙,使用碳酸钙、磷酸钙等钙盐以及蛋壳、鱼骨粉等天然钙制剂。然而,它们未必能称作适合经口摄取的原材料。对于轻微运动而言,认为慢跑或散步等较佳,但体弱的情况下即便是轻微的运动也非常麻烦,况且卧床的老人更是几乎无法运动。从补充活化维生素D3的观点来看,日光浴是很好的,但仅靠日光浴是不够的。药物投与中使用1α-羟基维生素D3、降钙素制剂等,已知其对骨质疏松症的治疗是有效的。然而,这些物质本身是药品,不能作为食品原材料使用。
本发明人等为了获得能够作为食品原材料使用的具有骨强化作用的物质,对存在于乳中的骨强化因子进行了持续的探索。结果发现,从乳清蛋白质的水溶性级分除去了乳清来源的盐而得的蛋白质和肽混合物具有骨强化作用(例如,参照专利文献1)。并且,发现对该蛋白质和肽混合物的水溶液进行乙醇处理、加热处理、加盐处理、超滤膜处理而得的级分具有促进成骨细胞增殖的作用和骨强化作用(例如,参照专利文献2、3)。另外,发现存在于乳中的碱性蛋白质具有促进成骨细胞增殖的作用、骨强化作用和防止骨吸收的作用(例如,参照专利文献4)。
专利文献1:日本专利3160862号公报
专利文献2:日本专利3092874号公报
专利文献3:日本特开平5-320066号公报
专利文献4:日本专利3112637号公报
发明内容
发明要解决的问题
本发明发现了一种能够作为食品原材料使用的、具有骨强化作用的乳蛋白质级分或乳蛋白质级分分解物,其课题在于提供一种含有该具有骨强化作用的乳蛋白质级分或乳蛋白质级分分解物的骨强化剂、以及配合有具有骨强化作用的乳蛋白质级分或乳蛋白质级分分解物的药品、饮食品或饲料。
用干解决问颗的方案
本发明人等尝试对新的骨强化原材料进行探索,结果发现获得了与以往的食品原材料相比显示出更强的骨强化作用的级分。并且,基于这些见解,以至于获得了含有该具有骨强化作用的乳蛋白质级分或乳蛋白质级分分解物的骨强化剂、以及配合有具有骨强化作用的乳蛋白质级分或乳蛋白质级分分解物的药品、饮食品和饲料。
本发明为包含下述方案的发明。
(A)涉及一种具有以下特性的乳蛋白质级分:
(1)其为乳来源;
(2)其为含有在十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)中分子量在6,000~150,000道尔顿范围内的蛋白质的级分;
(3)构成氨基酸的组成中含有12~14重量%的碱性氨基酸、且碱性氨基酸/酸性氨基酸的比在0.5~0.7的范围内;
(4)具有成骨细胞的钙化促进作用。
(B)一种乳蛋白质级分分解物,其通过用蛋白酶将前述乳蛋白质级分分解而获得。
(C)一种骨强化剂,其含有(A)所述的乳蛋白质级分或(B)所述的乳蛋白质级分分解物。
(D)一种骨强化用药品,其配合有(A)所述的乳蛋白质级分或(B)所述的乳蛋白质级分分解物。
(E)一种骨强化用饮食品,其配合有(A)所述的乳蛋白质级分或(B)所述的乳蛋白质级分分解物。
(F)一种骨强化用饲料,其配合有(A)所述的乳蛋白质级分或(B)所述的乳蛋白质级分分解物。
发明的效果
本发明的以具有骨强化作用的乳蛋白质级分或乳蛋白质级分分解物为有效成分的骨强化剂、以及含有具有骨强化作用的乳蛋白质级分或乳蛋白质级分分解物的骨强化用药品、饮食品和饲料,通过经口摄取,可在体内促进成骨细胞的钙化。
因此,本发明的以具有骨强化作用的乳蛋白质级分或乳蛋白质级分分解物为有效成分的骨强化剂、以及配合有具有骨强化作用的乳蛋白质级分或乳蛋白质级分分解物的骨强化用药品、饮食品和饲料,由于可在人或动物的生物体内促进成骨细胞的钙化,因而具有骨强化作用,且对于抑制骨质疏松症等骨量减少有效。
附图说明
图1所示为乳来源的蛋白质级分的钙化促进效果的说明图。(试验例1)。
图2所示为对骨质疏松症大鼠投与本发明的实施例1中获得的乳蛋白质级分时大腿骨骨强度增强效果的图(试验例2)。
具体实施方式
本发明涉及以具有骨强化作用的乳蛋白质级分或乳蛋白质级分分解物为有效成分的骨强化剂、以及配合有具有骨强化作用的乳蛋白质级分或乳蛋白质级分分解物的骨强化用药品、饮食品和饲料。
本发明的这种具有骨强化作用的乳蛋白质级分例如能够通过如下方式获得:使脱脂乳或乳清等乳原料与阳离子交换树脂接触,在用去离子水清洗后,用0.2M的食盐洗脱液使吸附于该树脂上的乳蛋白质洗脱从而获得。另外,作为盐类,除了食盐以外可以使用钾盐、铵盐、磷酸盐、醋酸盐、碳酸盐等,通过将清洗液的离子强度适当调整为0.05以下、将洗脱溶液的离子强度适当调整为0.15~0.25,能够获得本发明的乳蛋白质级分。另外,还可以通过将该洗脱级分回收,利用反渗透(RO)膜或电渗析(ED)法等进行脱盐和浓缩,并根据需要进行干燥来获得。作为反渗透(RO)膜,可以列举出Desal-3(Desalination公司制)、HR-95(Dow Danmark公司制)、NTR-729HF(日东电工公司制)等。另外,电渗析(ED)装置可以列举出YuasaIonics Inc.或日本炼水公司制的电渗析装置。
另外,作为获得乳来源的蛋白质级分的方法,已知有如下方法:在使乳或乳来源的原料与阳离子交换体接触后,用pH超过5、且离子强度超过0.5的洗脱液使吸附于该阳离子交换体上的碱性蛋白质级分洗脱,从而获得乳来源的蛋白质级分的方法〔日本特开平5-202098号公报〕;使用海藻酸凝胶而获得的方法(日本特开昭61-246198号公报);使用无机多孔性颗粒从乳清中获得的方法(日本特开平1-86839号公报);使用硫酸化酯化合物从乳中获得的方法(日本特开昭63-255300号公报)等。本发明的具有骨强化作用的乳来源的蛋白质级分可以使用通过这样的方法获得的蛋白质级分。
这样回收的乳蛋白质级分通常可以通过冷冻干燥等使其粉末化后使用。
本发明中使用的具有骨强化作用的乳蛋白质级分优选其构成氨基酸(constitutional amino acid)的组成中含有12~14重量%的碱性氨基酸、且碱性氨基酸/酸性氨基酸的比在0.5~0.7的范围内。若在该范围之外的话,无法发挥本申请发明的效果。另外,本发明品为分子量在6,000~150,000道尔顿的多种蛋白质的混合物,等电点分布于6~11的较宽范围。
乳蛋白质级分分解物具有与乳蛋白质级分同样的氨基酸组成,例如,可以通过使胃蛋白酶、胰蛋白酶、胰凝乳蛋白酶等蛋白酶作用于通过上述方法获得的乳蛋白质级分,进而根据需要用胰酶等蛋白酶作用,从而能够获得平均分子量在4,000以下的分解物。另外,乳蛋白质级分分解物通常可以通过冷冻干燥等粉末化后使用。
作为成为本发明的具有骨强化作用的乳蛋白质级分的供给源的乳或乳来源的原料,可以使用牛乳、人乳、山羊乳、羊乳等乳,可以直接使用这些乳,或者也可以使用这些乳的还元乳、脱脂乳、乳清等。
在投与本发明的骨强化剂时,可以将作为有效成分的具有骨强化作用的乳蛋白质级分或乳蛋白质级分分解物以其原本的状态使用,也可以根据常规方法制剂为粉末剂、颗粒剂、片剂、胶囊剂、饮用剂等而使用。此外,还可以将这些乳蛋白质级分或乳蛋白质级分分解物直接或者制剂化后,将其配合到营养剂或饮食品等中,从而谋求骨强化。另外,本发明的乳蛋白质级分或乳蛋白质级分分解物对于热比较稳定,因而还能够在通常进行的加热杀菌条件下加热杀菌。
本发明中,为了发挥骨强化作用,可以考虑体重、性别和年龄等而适当确定配合量,通常在成人的情况下,可以将本发明品即乳蛋白质级分或乳蛋白质级分分解物的配合量等调整为每天摄取1~50mg。这样,本发明品在低用量下也具有效果。这样,通过经口摄取将本发明的具有骨强化作用的成分制成的骨强化剂或者配合有其的药品、饮食品和饲料,从而发挥骨强化作用。
以下,示出参考例、实施例和试验例来详细说明本发明,但它们仅仅列举出本发明的实施方式,本发明并不受这些例子的任何限制。
[参考例1]
根据以下方法(参照日本专利第3112637号),调制可确认骨强化效果的市售乳蛋白质级分。
用去离子水充分清洗填充有0.5升作为阳离子交换树脂的磺化Chitopearl(富士纺绩制)的直径10cm的柱。以流速100ml/min使50升未杀菌脱脂乳通过该柱后,用去离子水充分清洗该柱,使含有0.95M氯化钠的0.05M磷酸缓冲液(pH7.0)2.5升通过该柱从而使吸附于树脂上的蛋白质洗脱。然后,通过反渗透(RO)膜处理对该洗脱液脱盐、浓缩后,进行冷冻干燥,获得粉末状的乳蛋白质级分。重复该操作2次,获得104g的蛋白质级分。该蛋白质级分的等电点为7.0~8.5,该蛋白质级分中所含的碱性氨基酸为17.8%。
实施例1
用去离子水充分清洗填充有0.5升作为阳离子交换树脂的磺化Chitopearl(富士纺绩制)的直径10cm的柱。以流速100ml/min使50升未杀菌脱脂乳通过该柱后,用去离子水充分清洗该柱,使含有0.15M氯化钠的0.05M磷酸缓冲液(pH7.0)2.5升通过该柱从而使吸附于树脂上的蛋白质洗脱。然后,通过反渗透(RO)膜处理对该洗脱液脱盐、浓缩后,进行冷冻干燥,获得粉末状的乳蛋白质级分。进行10次该操作,获得24.2g的乳蛋白质级分。该级分的分子量为6,000~150,000道尔顿、等电点为6.0~11.0,该乳蛋白质级分中所含的构成氨基酸中,碱性氨基酸为12~14%。另外,碱性氨基酸/酸性氨基酸的比为0.5~0.7。
实施例2
用去离子水充分清洗填充有0.5升作为阳离子交换树脂的磺化Chitopearl(富士纺绩制)的直径10cm的柱。以流速100ml/min使50升未杀菌脱脂乳通过该柱后,用含有0.05M氯化钠的0.05M磷酸缓冲液(pH7.0)充分清洗该柱,使含有0.25M氯化钠的0.05M磷酸缓冲液(pH7.0)2.5升通过该柱从而使吸附于树脂上的蛋白质洗脱。然后,通过反渗透(RO)膜处理对该洗脱液脱盐、浓缩后,进行冷冻干燥,获得粉末状的乳蛋白质级分。进行5次该操作,获得12.8g的乳蛋白质级分。该级分的分子量为6,000~150,000道尔顿、等电点为6.0~11.0,该乳蛋白质级分中所含的构成氨基酸中,碱性氨基酸为12~14%。另外,碱性氨基酸/酸性氨基酸的比为0.5~0.7。
实施例3
将实施例1中得到的乳蛋白质级分24.2g溶解于10升蒸馏水中后,以2%的浓度添加胃蛋白酶(关东化学制),在37℃下边搅拌1小时边进行水解。接着,用氢氧化钠溶液中和至pH6.8后,添加1%胰酶(Sigma公司制),在37℃下反应2小时。反应后,在80℃下加热处理10分钟而使酶失活,然后获得乳蛋白质级分分解物23.1g。
实施例4
将实施例2中得到的乳蛋白质级分12.8g溶解于8升蒸馏水中后,以2%的浓度添加胰蛋白酶(关东化学制),在37℃下边搅拌1小时边进行水解。接着,用氢氧化钠溶液中和至pH6.6后,添加1%胰酶(Sigma公司制),在37℃下反应2小时。反应后,在80℃下加热处理10分钟而使酶失活,然后获得乳蛋白质级分分解物11.7g。
[试验例1]
关于参考例1和实施例1中得到的各蛋白质级分,根据Danjo等的方法[Danjo.A.et al.,Biochem.Biophys.Res.Commun.,vol.360,pp.199-204,2007]进行了研究。即,以5×103cell/cm2的细胞密度将小鼠颅盖来源的成骨细胞样细胞MC3T3-E1悬浮于αMEM+10%FBS培养基中,接种于24孔的培养皿。静置12小时后,更换成钙化诱导培养基(αMEM+10%FBS 50μg/ml+5mM β-磷酸甘油),同时分别以10-10~10-5M的浓度添加参考例1中得到的乳蛋白质和实施例1中得到的本发明品。每3天更换一次培养基,连续培养28天。
在培养的第14、21、28天,用含有4%多聚甲醛的0.1M磷酸缓冲液(pH7.4)在室温下固定10分钟。用0.01M PBS清洗后,用2%茜素红S水溶液在室温下反应15分钟,进行钙染色。为了使作为骨的无机成分之一的磷酸染色,在固定后用蒸馏水清洗,并用5%硝酸银水溶液在室温下反应1小时,进行冯库萨(vonKossa)染色。在钙化小结节内的钙离子量的定量中使用了Calcium C试剂盒(和光纯药工业公司制)。
图1所示为培养第28天的钙浓度测定结果。将在仅未添加试样的培养基中培养的组作为对照。
关于实施例1中得到的本发明品,确认到具有用量依赖性地促进成骨细胞钙化的特性。参考例1中得到的乳蛋白质虽然也确认到了同样的作用,但可知与本发明品相比其作用较弱。
[试验例2]
关于参考例1和实施例1中得到的各蛋白质级分,通过动物实验研究了骨强化作用。动物实验中使用了4周龄的Wistar系雌性大鼠。在1周的预备饲育后,实施卵巢摘除手术,然后以钙缺乏饲料饲育5周并供于动物实验。另外,摘除了卵巢、并以钙缺乏饲料饲育了5周的大鼠明显处于骨质疏松症的状态。将产生了该骨质疏松症状态的大鼠以1组6只分别分配到下述5个实验组:未添加乳蛋白质级分的对照组(A组)、添加了0.5重量%的参考例1中得到的乳蛋白质级分的投与组(B组)、添加了0.1重量%的实施例1中得到的乳蛋白质级分的投与组(C组)、添加了0.5重量%的实施例1中得到的乳蛋白质级分的投与组(D组)、添加了1.0重量%的实施例1中得到的乳蛋白质级分的投与组(E组),分别用表1所示的试验饲料饲育4个月。另外,用酪蛋白调整各试验饲料的氮含量,使其具有相同的氮含量(17.06%)。另外,对于各试验饲料,在每100g中配合300mg钙、230mg磷和50mg镁。
表1
(重量%)
饲育4个月后,摘除各试验组大鼠的两侧大腿骨,用骨断裂力测定装置(Rheometer Max型号RX-1600,Aitecno Inc.制)测定骨强度。其结果如图2所示。由此,与对照组(A组:未添加乳蛋白质级分)相比,在参考例1中得到的乳蛋白质级分投与组(B组)、实施例1中得到的乳蛋白质级分投与组(C组、D组、E组)中大腿骨断裂应力显示出较高的值。另外,大腿骨断裂应力随着实施例1中得到的乳蛋白质级分的浓度增加而显示出较高的值。参考例1中得到的乳蛋白质级分虽然也确认到了增加大腿骨断裂应力的作用,但可知实施例1中得到的本发明品的该作用更强。
另外,虽然没有示出实验结果,但在使用了实施例3和实施例4中得到的乳蛋白质级分分解物的情况下也确认到了同样的效果。
实施例5
在100mg实施例1中得到的乳蛋白质级分中添加93.4g水合晶体葡萄糖、5g碳酸钙、1g糖酯、0.5g香料,混和,然后压片而制成片状,制造本发明的骨强化剂。
实施例6
以表2所示的组成混合各成分并制成生面团,成形后进行烘焙,制造用于预防和改善骨质疏松症等各种骨疾病的饼干。
表2
实施例7
制造表3所示的组成的骨强化用果汁饮料。
表3
实施例8
根据表4所示的配方混合原料,制造骨强化用狗饲育饲料。
表4
实施例9
以表5所示的组成混合各成分,加压成形,调制配合有实施例3中得到的乳蛋白质级分分解物的骨强化用片剂。
表5
实施例10
以表6所示的组成混合各成分,在乳化温度85℃下乳化,调制配合有实施例4中得到的乳蛋白质级分分解物的骨强化用加工干酪。
表6
产业上的可利用性
本发明的乳蛋白质级分具有骨强化作用,因而作为以骨强化等为目的的骨强化剂对于骨疾病的预防或治疗非常有用。另外,本发明品的安全性很高,能够制成配合有该骨强化剂的骨疾病的预防或治疗用药品、饮食品和饲料使用。
Claims (6)
1.一种乳蛋白质级分,其具有以下(1)~(4)的特性:
(1)其为乳来源;
(2)其为含有在十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)中分子量在6,000~150,000道尔顿范围内的蛋白质的级分;
(3)构成氨基酸的组成中含有12~14重量%的碱性氨基酸、且碱性氨基酸/酸性氨基酸的比在0.5~0.7的范围内;
(4)具有成骨细胞的钙化促进作用。
2.一种乳蛋白质级分分解物,其通过用蛋白酶将权利要求1所述的乳蛋白质级分分解而获得。
3.一种骨强化剂,其含有权利要求1所述的乳蛋白质级分或权利要求2所述的乳蛋白质级分分解物。
4.一种骨强化用药品,其配合有权利要求1所述的乳蛋白质级分或权利要求2所述的乳蛋白质级分分解物。
5.一种骨强化用饮食品,其配合有权利要求1所述的乳蛋白质级分或权利要求2所述的乳蛋白质级分分解物。
6.一种骨强化用饲料,其配合有权利要求1所述的乳蛋白质级分或权利要求2所述的乳蛋白质级分分解物。
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