CN110946989A - 一种含鲟鱼蛋白肽的组合物的应用 - Google Patents
一种含鲟鱼蛋白肽的组合物的应用 Download PDFInfo
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- CN110946989A CN110946989A CN201911010470.5A CN201911010470A CN110946989A CN 110946989 A CN110946989 A CN 110946989A CN 201911010470 A CN201911010470 A CN 201911010470A CN 110946989 A CN110946989 A CN 110946989A
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Abstract
本发明公开了一种组合物及其在制备抗心血管疾病中的应用。所述组合物包括鲟鱼蛋白肽、纳豆粉、茯苓多糖、以及归经包括脾经的中药提取物;其中所述鲟鱼蛋白肽与所述纳豆粉的质量比例在1~1.5:1之间,所述鲟鱼蛋白肽与所述茯苓多糖的质量比例在1.8~2.2:1之间;所述归经包括脾经的中药提取物与茯苓多糖的质量比例在1.5~2.5:1之间;所述组合物中含有5000~10000IU/g纳豆激酶。本发明提供的组合物(商品名“鲟纳茯精华片”)具有明显的降低血脂和溶解血栓的作用,采用食品或已确认的药食同源药品,能满足食品的安全性要求,适合长期使用,能应用于制备以预防为主、防治结合的心血管疾病防治药物。
Description
技术领域
本发明属于生物医药领域,更具体地,涉及一种含鲟鱼蛋白肽的组合物的应用。
背景技术
色斑,是指和周围颜色不同的斑点。包括雀斑、黑斑、黄褐斑和老年斑等,属色素障碍性皮肤病。由于皮肤黑色素的增加而形成的一种常见面部呈褐色或黑色素沉着性、损容性的皮肤疾病,多发于面颊和前额部位,日晒后加重,多见于女性,与妊娠、长期月经紊乱有关。色斑确切的形成原因并不明确。因此缺乏有效的预防和治疗手段。
目前对于色斑的治疗和预防方案,包括防晒、和激光治疗、药物治疗等。然而防晒只能一定程度的减少黑色素产生,并不能避免产生色斑,同时对已经产生的色斑几乎没有作用;激光治疗,恢复时间短、效果明显,然而成本较高,并且容易复发,效果难以持续;药物治疗对某些因激素问题导致的色斑有一定疗效,然而色斑成因复杂,因此药物治疗的效果并不稳定。需要开发更多的具有祛斑作用的内服食品、药品、保健品等。
发明内容
针对现有技术的以上缺陷或改进需求,本发明提供了一种含鲟鱼蛋白肽的组合物的应用,其目的在于,实践发现本发明提供的含鲟鱼蛋白肽的组合物具有显著的祛斑作用,能应用于制备内服食品、药品、以及保健品。
为实现上述目的,按照本发明的一个方面,提供了一种含鲟鱼蛋白肽的组合物的应用,所述含鲟鱼蛋白肽的组合物包括鲟鱼蛋白肽、纳豆粉、茯苓多糖、以及归经包括脾经的中药提取物;其中所述鲟鱼蛋白肽与所述纳豆粉的质量比例在1~1.5:1之间,所述鲟鱼蛋白肽与所述茯苓多糖的质量比例在1.8~2.2:1之间;所述归经包括脾经的中药提取物与茯苓多糖的质量比例在1.5~2.5:1之间;所述组合物中含有5000~10000IU/g纳豆激酶,其应用于制备具有祛斑作用的内服食品、药品或保健品。
优选地,所述应用,其所述鲟鱼蛋白肽含有1.2~1.5mg/g的金属硫蛋白肽。
优选地,所述应用,其所述鲟鱼蛋白肽按照如下方法制备:
1)将鲟鱼洗净、切成块状,然后绞碎,加入其重量1-3倍的水,升温至90℃以上保持10-20min,然后在2-2.5MPa的压力下进行汽爆处理3-5min;
2)步骤1)得到的物料冷却至60℃以下,加入盐酸溶液调pH至1.5-5,再加入鲟鱼质量0.2~0.3%的胃蛋白酶原进行酶解反应2至5小时;
3)步骤2)得到的物料加入碱溶液调节pH至7.5至8,再加入鲟鱼质量0.1~0.2%的胰蛋白酶进行酶解反应1至3小时;
3)步骤2)得到的物料升温至90℃以上保持10-20min,再冷却至65℃以下,进行脱色、脱盐、除菌过滤、滤液浓缩,最后经消毒干燥后即可得到蛋白肽粉体;
其平均分子量在250Da以下。
优选地,所述应用,其所述纳豆粉为采用枯草杆菌对黑豆进行发酵的制得的纳豆粉末。
优选地,所述应用,其所述归经包括脾经的中药提取物为火麻仁提取物、以及郁李仁提取物的一种或两种的组合,优选所述归经包括脾经的中药提取物为火麻仁提取物和郁李仁提取物按照质量比例1:0.8~1.2的组合。
优选地,所述应用,其所述茯苓多糖为取代度在0.69至0.73的羧甲基茯苓多糖。
优选地,所述应用,其所述组合物具有肠溶包衣。
优选地,所述应用,其应用于制备具有祛除老年斑、黄褐斑作用的内服食品、药品、保健品。
总体而言,通过本发明所构思的以上技术方案与现有技术相比,能够取得下列有益效果:
本发明提供的组合物有效复配了鲟鱼蛋白肽、纳豆粉、茯苓多糖、以及归经包括脾经的中药提取物,人群实验显现出具有统计显著性的祛斑作用,能应用于制备具有祛斑作用的内服药品、食品、保健品。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白,以下结合实施例,对本发明进行进一步详细说明。应当理解,此处所描述的具体实施例仅仅用以解释本发明,并不用于限定本发明。此外,下面所描述的本发明各个实施方式中所涉及到的技术特征只要彼此之间未构成冲突就可以相互组合。
本发明提供的组合物,包括鲟鱼蛋白肽、纳豆粉、茯苓多糖、以及归经包括脾经的中药提取物;其中所述鲟鱼蛋白肽与所述纳豆粉的质量比例在1~1.5:1之间,所述鲟鱼蛋白肽与所述茯苓多糖的质量比例在1.8~2.2:1之间;所述归经包括脾经的中药提取物与茯苓多糖的质量比例在1.5~2.5:1之间;所述组合物中含有5000~10000IU/g纳豆激酶。
所述鲟鱼蛋白肽含有1.2~1.5mg/g的金属硫蛋白肽;所述鲟鱼蛋白肽按照如下方法制备:
1)将鲟鱼洗净、切成块状,然后绞碎,加入其重量1-3倍的水,升温至90℃以上保持10-20min,然后在2-2.5MPa的压力下进行汽爆处理3-5min;
2)步骤1)得到的物料冷却至60℃以下,加入盐酸溶液调pH至1.5-5,再加入鲟鱼质量0.2~0.3%的胃蛋白酶原进行酶解反应2至5小时;
3)步骤2)得到的物料加入碱溶液调节pH至7.5至8,再加入鲟鱼质量0.1~0.2%的胰蛋白酶进行酶解反应1至3小时;
4)步骤3)得到的物料升温至90℃以上保持10-20min,再冷却至65℃以下,进行脱色、脱盐、除菌过滤、滤液浓缩,最后经消毒干燥后即可得到蛋白肽粉体。
平均分子量在250Da以下。
所述纳豆粉为使用菌种Bacillus subtilis QK02枯草杆菌对黑豆进行发酵的制得的纳豆粉末,菌种Bacillus subtilis QK02由中国典型培养物保藏中心保藏,保藏编号为CCTCC NO:M203078,所述纳豆粉按照如下方法制备:
(1)Bacillus subtilis QK02种子液的培养:
种子液是由斜面菌株经摇瓶培养制备。培养条件为:以LB培养基为种子培养基,pH为7.2,三角瓶装液量为25mL/250mL,培养温度37℃,摇床转速为180r/min,培养周期为16h。
(2)原料预处理:
取籽粒饱满的黑豆进行除杂清洗后,每10kg加入30L营养液浸泡16h;将浸泡过的黑豆沥干液体后放入蒸煮设备中于0.1MPa、121℃蒸煮40min;所述营养液成分为:乳糖10g/L、胰蛋白胨20g/L、Na2HPO4·12H2O 5g/L、NaH2PO4·2H2O 1g/L、MgSO4·7H2O 0.5g/L、以及CaCl20.2g/L,调节pH 7.2。
(3)固体发酵:
将蒸煮后的黑豆原料自然冷却至50℃后于无菌条件下按黑豆重量的10%接种Bacillus subtilis QK02种子液,然后于发酵设备中进行发酵,优选控制物料厚度为3cm,温度为37℃,湿度为75%,发酵时间为24h。
(4)冷冻干燥:
将固体发酵后的物料置于-80℃超低温冰箱中冻结,然后放入预冷的真空冷冻干燥机中,于真空度为10Pa,冷阱温度-50℃条件下干燥24h,粉碎得到纳豆粉。
所述茯苓多糖优选为取代度在0.69至0.73的羧甲基茯苓多糖,按照如下方法制备:
第一步:将干燥过的茯苓菌核进行粉碎过80或100目筛,得到粒度均匀的茯苓粉;
第二步:将第一步获得的茯苓粉以60Co-γ为辐照源,对其进行辐照,辐照剂量为9kGy~27kGy;
第三步:以辐照后的茯苓粉作为羧甲基化反应底物,加入乙醇溶液进行溶胀;所述乙醇溶液浓度为85%,溶胀料液比为酒精溶液与茯苓粉的料液比在5~7∶1之间。
第四步:溶胀后加入氢氧化钠进行碱化反应,反应结束后,冷却;碱化反应中所述的氢氧化钠加入的量为茯苓粉质量的0.3~0.4倍;加入氢氧化钠之后进行搅拌,碱化反应0.5~1h,反应温度为40~60℃;反应结束后,应进行冷却,冷却至温度10~30℃;
第五步:加入氯乙酸和过量的氢氧化钠与茯苓全粉进行羧甲基化反应,得到所述取代度在0.69至0.73的羧甲基茯苓多糖。具体地,羧甲基化反应中所述氯乙酸的加入量为茯苓粉质量的0.8~1.2倍,使茯苓粉和氯乙酸在过量的氢氧化钠下进行醚化反应3~4h,反应温度为50~70℃,反应完毕后,用冰乙酸调至pH至5.0~6.0,用体积分数80~90%的乙醇洗涤3~5次,除去多余氯离子,并洗至白色或淡黄色,在50℃温度条件下干燥。
所述归经包括脾经的中药提取物为火麻仁提取物、以及郁李仁提取物的一种或两种的组合,优选所述归经包括脾经的中药提取物为火麻仁提取物和郁李仁提取物按照质量比例1:0.8~1.2的组合。
所述火麻仁提取物为桑科植物大麻Cannabis sativa L.的干燥成熟种子的黄棕色粉末状提取物。
本发明提供的含鲟鱼蛋白肽的组合物,
实验证实,本发明提供的组合物(商品名“鲟纳茯精华片(嫦娥II号)”)对于降低血液黏度效果明显,成分安全适合长期使用,因此能用于防治心血管疾病药物的制备,尤其是应用于溶血栓药物的制备,一方面对已经形成的血栓具备溶解作用,同时降低血栓形成风险;同时其具有明显降低血脂的作用,能用于降血脂药物的制备。
以下为实施例:
实施例中鲟鱼蛋白肽按照如下方法制备:
1)将鲟鱼洗净、切成块状,然后绞碎,加入其重量1-3倍的水,升温至90℃以上保持20min,然后在2.5MPa的压力下进行汽爆处理3min;
2)步骤1)得到的物料冷却至60℃以下,加入盐酸溶液调pH至1.5-5,再加入鲟鱼质量0.3%的胃蛋白酶原进行酶解反应5小时;
3)步骤2)得到的物料加入碱溶液调节pH至7.5至8,再加入鲟鱼质量0.1%的胰蛋白酶进行酶解反应2小时;
4)步骤3)得到的物料升温至90℃以上保持20min,再冷却至65℃以下,进行脱色、脱盐、除菌过滤、滤液浓缩,最后经消毒干燥后即可得到蛋白肽粉体。脱色剂采用活性炭和硅藻土按质量比2:1混合得到,脱色剂的加入量为鲟鱼质量的0.2-0.3%,脱色时间为30min,温度控制在60-70℃;采用枯草芽孢杆菌发酵进行脱腥处理;采用反渗透脱盐处理,所述反渗透脱盐处理参数:压力1MP,温度25℃,脱盐率70%,醋酸纤维素膜;再采用微滤膜进行除菌过滤;采用纳滤膜对滤液浓缩,最后经瞬时高温消毒和真空冷冻干燥后即可得到蛋白肽粉体。
所述鲟鱼蛋白肽,含有1.5mg/g的金属硫蛋白肽;委托北京中科光析化工技术研究所(食品实验室)进行多肽含量及分子量分布检测结果显示平均分子量为243。
实施例中纳豆粉按照如下方法制备:
(1)Bacillus subtilis QK02种子液的培养:
种子液是由斜面菌株经摇瓶培养制备。培养条件为:以LB培养基为种子培养基,pH为7.2,三角瓶装液量为25mL/250mL,培养温度37℃,摇床转速为180r/min,培养周期为16h。
(2)原料预处理:
取籽粒饱满的黑豆进行除杂清洗后,每10kg加入30L营养液浸泡16h;将浸泡过的黑豆沥干液体后放入蒸煮设备中于0.1MPa、121℃蒸煮40min;所述营养液成分为:乳糖10g/L、胰蛋白胨20g/L、Na2HPO4·12H2O 5g/L、NaH2PO4·2H2O 1g/L、MgSO4·7H2O 0.5g/L、以及CaCl20.2g/L,调节pH7.2。
(3)固体发酵:
将蒸煮后的黑豆原料自然冷却至50℃后于无菌条件下按黑豆重量的10%接种Bacillus subtilis QK02种子液,然后于发酵设备中进行发酵,优选控制物料厚度为3cm,温度为37℃,湿度为75%,发酵时间为24h。
(4)冷冻干燥:
将固体发酵后的物料置于-80℃超低温冰箱中冻结,然后放入预冷的真空冷冻干燥机中,于真空度为10Pa,冷阱温度-50℃条件下干燥24h,粉碎得到纳豆粉。
实施例中茯苓多糖照如下方法制备:
取1kg过100目筛的茯苓粉,以60Co-γ为辐照源辐照24h,加入5.0L 85%乙醇在室温下搅拌溶胀30min,再加入0.3~0.5kg的氢氧化钠(用少量乙醇溶解)在50℃下搅拌碱化0.5~1.0h。碱化后,冷却反应体系至30℃以下,然后再加入0.3~0.5kg氢氧化钠(用少量乙醇溶解)和0.8~1.0kg氯乙酸,在50~70℃温度条件下醚化3.0h。反应完毕后,用体积分数80%的乙醇洗涤4次,除去多余氯离子。在40~50℃下干燥7~10h,过40目筛,得到羧甲基茯苓多糖粉。
羧甲基取代度测定:
D·S按照灰碱法测定,测定方法如下:将已充分干燥后的改性产品连同瓷埚锅放进高温炉,700±25℃灼烧30min,冷却后先用少量水润湿含有焦粒的灰白粉末,然后洗移至烧杯中(总量不超150mL),加入甲基红指示剂3滴,用0.05mol/L H2SO4滴到显红色后,再从滴定管放入10mL,记下H2SO4总用量a,将溶液放在电炉上加热,使之轻轻沸腾10min,不必冷却,即用0.1mol/L的NaOH溶液滴定至变黄色为止,记下其用量b。计算公式:
取代度(D·S)=0.162B/(l-0.08B)
B=(a*Nl-b*N2)/W
a一硫酸标准溶液用量(mL)
b一氢氧化钠标准溶液用量(mL)
Nl一硫酸标准溶液当量浓度(0.05mol/L)
N2一氢氧化钠标准溶液当量浓度(0.l mol/L)
W一样品重量(g)
本发明制备的CMP的取代度(D·S)=0.6902
实施例中火麻仁提取物可以通过市售购买获得,也可以采用本领域熟知的提取方法提取获得,所述的提取方法例如是浸泡提取、加热提取、回流提取、超声提取、逆流提取、超临界萃取中的一种或两种以上的组合。
实施例中郁李仁提取物可以通过市售购买获得,也可以采用本领域熟知的提取方法提取获得,所述的提取方法例如是浸泡提取、加热提取、回流提取、超声提取、逆流提取、超临界萃取中的一种或两种以上的组合。
实施例提供的组合物成分配比如下表:
实施例6
按照质量份数,将实施例1~5中的组合物100份与0.5麦芽糊精、0.5份木糖醇、0.3份硬脂酸镁压制成片,每片0.5g,包裹肠溶包衣,其中实施例5制得商品名为“鲟纳茯精华片”的保健食品。肠溶包衣材料:苯二甲酸醋酸纤维素8.0g,聚对苯二甲酸乙二醇酯2.4ml,乙酸乙酯33.0ml和乙酸异丙酯33.6ml,在氮气保护下至少用1层。
人群实验:
将实施例5制备的肠溶包衣片,一日2次,每次2片,三个月为一疗程,提供给换自评有祛斑需求的人群受试者,分别为雀斑组、晒斑组、黄褐斑组、老年斑组,各组100例,共计400例。各组受试者在测试开始前采用武汉嫦娥医学抗衰机器人股份有限公司研发的皮肤分析仪(嫦娥抗衰豪华版皮肤解码机器人),记录受试者皮肤表面斑点数据,然后按照要求使用实施例5制备的肠溶包衣片,
2个疗程后根据感官评价和仪器评价的结果统计有效率。
感官评价:受试者自我评价斑点淡化情况,分为:不明显、明显、非常明显。结果统计表如下:
表1感官评价结果
不明显 | 明显 | 非常明显 | |
雀斑组 | 63 | 27 | 10 |
晒斑组 | 51 | 31 | 19 |
黄褐斑组 | 20 | 58 | 22 |
老年斑组 | 19 | 45 | 36 |
仪器评价:斑点区域由皮肤分析仪自动勾勒,以斑点参数(斑点区域占全脸面积百分比)为评价依据,将受试者半点淡化情况分为:不明显(半点参数提高或斑点参数降低在2%以内),明显(斑点参数降低10%以内),非常明显(斑点参数降低10%以上)。结果统计表如下:
表2仪器评价结果
不明显 | 明显 | 非常明显 | |
雀斑组 | 40 | 51 | 9 |
晒斑组 | 49 | 40 | 11 |
黄褐斑组 | 19 | 60 | 21 |
老年斑组 | 18 | 48 | 33 |
对比表1和表2可知,感官评价结果与仪器评价结果基本一致,说明结果的可靠性。实施例5制备的肠溶包衣片对各类斑点均能起到一定的效果,其中对于活性斑点雀斑、晒斑效果不明显,且其自然淡化效果未评价;对于黄褐斑、老年斑等定性斑点,具有较为明显淡化效果。
本领域的技术人员容易理解,以上所述仅为本发明的较佳实施例而已,并不用以限制本发明,凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等,均应包含在本发明的保护范围之内。
Claims (8)
1.一种含鲟鱼蛋白肽的组合物的应用,其特征在于,所述含鲟鱼蛋白肽的组合物包括鲟鱼蛋白肽、纳豆粉、茯苓多糖、以及归经包括脾经的中药提取物;其中所述鲟鱼蛋白肽与所述纳豆粉的质量比例在1~1.5:1之间,所述鲟鱼蛋白肽与所述茯苓多糖的质量比例在1.8~2.2:1之间;所述归经包括脾经的中药提取物与茯苓多糖的质量比例在1.5~2.5:1之间;所述组合物中含有5000~10000IU/g纳豆激酶,其应用于制备具有祛斑作用的内服食品、药品或保健品。
2.如权利要求1所述的应用,其特征在于,所述鲟鱼蛋白肽含有1.2~1.5mg/g的金属硫蛋白肽。
3.如权利要求1所述的应用,其特征在于,所述鲟鱼蛋白肽按照如下方法制备:
1)将鲟鱼洗净、切成块状,然后绞碎,加入其重量1-3倍的水,升温至90℃以上保持10-20min,然后在2-2.5MPa的压力下进行汽爆处理3-5min;
2)步骤1)得到的物料冷却至60℃以下,加入盐酸溶液调pH至1.5-5,再加入鲟鱼质量0.2~0.3%的胃蛋白酶原进行酶解反应2至5小时;
3)步骤2)得到的物料加入碱溶液调节pH至7.5至8,再加入鲟鱼质量0.1~0.2%的胰蛋白酶进行酶解反应1至3小时;
3)步骤2)得到的物料升温至90℃以上保持10-20min,再冷却至65℃以下,进行脱色、脱盐、除菌过滤、滤液浓缩,最后经消毒干燥后即可得到蛋白肽粉体;
其平均分子量在250Da以下。
4.如权利要求3所述的应用,其特征在于,所述纳豆粉为采用枯草杆菌对黑豆进行发酵的制得的纳豆粉末。
5.如权利要求1所述的应用,其特征在于,所述归经包括脾经的中药提取物为火麻仁提取物、以及郁李仁提取物的一种或两种的组合,优选所述归经包括脾经的中药提取物为火麻仁提取物和郁李仁提取物按照质量比例1:0.8~1.2的组合。
6.如权利要求1所述的应用,其特征在于,所述茯苓多糖为取代度在0.69至0.73的羧甲基茯苓多糖。
7.如权利要求1所述的应用,其特征在于,所述组合物具有肠溶包衣。
8.如权利要求1所述的应用,其特征在于,应用于制备具有祛除老年斑、黄褐斑作用的内服食品、药品、保健品。
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