CN105901738A - 一种发酵葡萄提取物及其制备方法与含其制剂 - Google Patents
一种发酵葡萄提取物及其制备方法与含其制剂 Download PDFInfo
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- CN105901738A CN105901738A CN201610436994.0A CN201610436994A CN105901738A CN 105901738 A CN105901738 A CN 105901738A CN 201610436994 A CN201610436994 A CN 201610436994A CN 105901738 A CN105901738 A CN 105901738A
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- ethanol
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Abstract
本发明涉及一种发酵葡萄提取物及其制备方法与含其制剂,该发酵葡萄提取物含有葡萄多酚和白藜芦醇。本发明是利用酵母菌发酵全葡萄的方法,将全葡萄葡中的多酚类物质提取出来,提取率显著提高。同时操作简单,成本低,适用于工业生产。实验证实,本发明提供的发酵葡萄提取物能显著提高小鼠体内抗氧化酶的活性,具有抗氧化的作用,效果优于现有技术。
Description
技术领域
本发明涉及一种发酵葡萄提取物及其制备方法与含其制剂,尤其是涉及一种利用全葡萄进行发酵来制备葡萄提取物的方法。
背景技术
葡萄多酚具有很强的抗氧化活性,在消炎、抗突变、抗肿瘤和抗心脑血管疾病方面具有显著功效,并能有效保护正常细胞不受损伤。其中含有的白藜芦醇在近几年,更是受到了广泛的关注。白藜芦醇是生物为抵抗环境压力(如气候恶劣、微生物侵染等)而产生的化学物质,具有显著的药理和生物学作用,如抗癌、保护神经、抗衰老、抗炎症以及延长寿命等。
葡萄是世界上产量最大的水果之一,无论是葡萄藤、葡萄叶、葡萄梗、葡萄皮以及葡萄籽中均含有大量的多酚类物质。目前,利用发酵全葡萄的方法,并在得到的发酵液以及滤渣中都进行提取葡萄多酚的方法还没有相关专利以及文献。
发明内容
本发明的目的在于克服现有制备工艺的不足,提供一种发酵葡萄提取物及其制备方法与含其制剂。本发明是利用酵母菌发酵全葡萄的方法,将全葡萄葡,包括葡萄藤、葡萄叶、葡萄梗、葡萄皮以及葡萄籽中的多酚类物质提取出来,提取率显著提高。同时操作简单,成本低,适用于工业生产。
本发明是通过以下技术方案实现的:
一种发酵葡萄提取物,含有葡萄多酚和白藜芦醇。
优选地,所述发酵葡萄提取物中葡萄多酚的含量≥30%,白藜芦醇的含量≥8%。
进一步优选地,所述发酵葡萄提取物中葡萄多酚的含量为30%-50%,白藜芦醇的含量为8%-20%。
本发明还提供上述发酵葡萄提取物的制备方法,包括以下步骤:
将葡萄发酵液过滤,得滤液A和滤渣;将滤渣干燥,粉碎,用乙醇提取,得到提取液;将所述滤液A和提取液合并,浓缩至干,得到粗提物;将所得粗提取物溶于体积比为1:1-1:2(优选体积比为2∶3)的乙醇与乙酸乙酯的混合液,过滤,得滤液B;将滤液B过大孔吸附树脂柱,以乙醇与乙酸乙酯体积比为7:3-9:1(优选体积比为4∶1)的洗脱液进行洗脱,收集洗脱液;减压浓缩,静置析晶,冷冻干燥,即得所述发酵葡萄提取物。
上述制备方法中,
优选地,用乙醇提取的方法包括加入滤渣干重8-15倍的60%-90%的乙醇,提取1-3次,每次1-4h,合并滤液,得到提取液。
优选地,滤液B过大孔吸附树脂柱的流速为1ml/min。
所述大孔树脂为ADS-F8、ADS-17、HPD-750、D-101、HPD-100、HPD-450或DA-201中的一种;优选为D-101。
优选地,将浓缩液在3-5℃条件下静置析晶。
优选地,所述葡萄发酵液是经酿酒活性干酵母发酵制得的。本发明研究发现,采用酿酒活性干酵母进行发酵比传统酿酒酵母所得提取物中葡萄多酚和白藜芦醇含量更高。
优选地,所述酿酒活性干酵母的用量为占葡萄原料重量的0.1-0.5‰,进一步优选为0.2-0.25‰。
优选地,葡萄原料为全葡萄原料,包括葡萄藤、葡萄叶、葡萄梗、葡萄皮、葡萄籽等。
优选地,所述发酵温度10-28℃,发酵7-15d。
具体地,上述发酵葡萄提取物的制备方法,包括以下步骤:
1)将全葡萄原料清洗破碎成浆,加入全葡萄重量0.2-0.25‰的酿酒活性干酵母,控制发酵温度10-28℃,发酵7-15d;
2)将发酵液过滤,得到滤液A;
3)将滤渣干燥,粉碎,加入滤渣干重8-15倍的60%-90%的乙醇,提取1-3次,每次1-4h,合并滤液,得到提取液;
4)将所述滤液A和提取液合并,浓缩至干,得到粗提物;
5)将所得粗提取物溶于体积比为1:1-1:2(优选体积比为2∶3)的乙醇与乙酸乙酯的混合液,过滤,得滤液B;
将滤液B过大孔吸附树脂柱,以乙醇与乙酸乙酯体积比为7:3-9:1(优选体积比为4∶1)的洗脱液进行洗脱,收集洗脱液;
6)将所得洗脱液,减压浓缩,将浓缩液在3-5℃条件下静置24-72h析晶,冷冻干燥,即得所述发酵葡萄提取物。
本发明所述酿酒活性干酵母可市售获得,例如购自安琪酵母股份有限公司。
本发明还包括按上述方法制备的发酵葡萄提取物。
本发明还提供了含上述发酵葡萄提取物的制剂,该制剂由发酵葡萄提取物和药学上可接受的载体或稀释剂组成。
所述药学上可接受的载体或稀释剂是指药学领域常规的药物载体,选自填充剂、粘合剂、崩解剂、润滑剂、助悬剂、润湿剂、溶剂、表面活性剂或矫味剂中的一种或几种。
所述填充剂选自淀粉、蔗糖、乳糖、甘露醇、山梨醇、木糖醇、微晶纤维素或葡萄糖等;
所述粘合剂选自纤维素衍生物、藻酸盐、淀粉、糊精、明胶或聚乙烯吡咯烷酮等;
所述崩解剂选自微晶纤维素、羧甲基淀粉钠、交联聚乙烯吡咯烷酮、低取代羟丙基纤维素或交联羧甲基纤维素钠;
所述润滑剂选自硬脂酸、聚乙二醇、碳酸钙、碳酸氢钠、微粉硅胶、滑石粉或硬脂酸镁;
所述助悬剂选自微粉硅胶、蜂蜡、纤维素、固态聚乙二醇;
所述润湿剂选自甘油、吐温-80、乙氧基氢化蓖麻油或卵磷脂;
所述溶剂选自乙醇、液态聚乙二醇、异丙醇、吐温-80、甘油、丙二醇或植物油,所述植物油选自大豆油、蓖麻油、花生油、调和油等;
所述表面活性剂选自十二烷基苯磺酸钠、硬脂酸、聚氧乙烯-聚氧丙烯共聚物、脂肪酸山梨坦或聚山梨酯(吐温)等;
所述矫味剂选自阿斯巴甜、蔗糖素、香精、柠檬酸或糖精钠。
所述制剂优选为片剂、胶囊剂、颗粒剂、滴丸剂或口服液等。
所述制剂可按本领域常规方法制备。
本发明还提供上述发酵葡萄提取物或含其制剂在制备抗氧化功能的保健食品中的应用。
本发明发酵葡萄提取物的制备方法,先将全葡萄破碎成浆发酵,再将发酵液以及滤渣都进行提取,以保证将葡萄藤、葡萄叶、葡萄皮、葡萄梗、葡萄籽、葡萄肉中存在的多酚都充分的提取出来。使得发酵葡萄提取物的提取率得到显著的提高。本发明采用酿酒活性干酵母进行发酵比传统酿酒酵母所得提取物中葡萄多酚和白藜芦醇含量更高,且还具有抗氧化等效果。本发明采用乙醇与乙酸乙酯进行萃取,结合大孔吸附树脂柱层析分离的方法,方法简便,大大提高了提取率。
本发明制备方法能够得到葡萄多酚和白藜芦醇类组分;具有一次性即可得到多种有效成分的优点。本发明通过对提取的关键参数进行严格控制,既保证了有效成分的溶出,又从多方面降低杂质量。本发明提供的发酵葡萄提取物,多种药效成分配伍,起协同作用,疗效更好。
本发明方法多酚类物质的提取率可达32.28mg/g,发酵葡萄提取物中葡萄多酚含量≥30%,白藜芦醇含量在≥8%。
本发明提供的制备方法工艺新颖,生产成本低,操作简单、节能、环保,适宜工业化生产。
实验证实,本发明提供的发酵葡萄提取物可显著提高小鼠体内抗氧化酶活性,具有抗氧化的作用,效果优于现有技术。
具体实施方式
以下实施例用于说明本发明,但不用来限制本发明的范围。实施例中未注明具体技术或条件者,按照本领域内的文献所描述的技术或条件,或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者,均为可通过正规渠道商购买得到的常规产品。以下活性干酵母(酿酒、面包)购自安琪酵母股份有限公司。
以下实施例葡萄多酚含量的检测方法为高效液相色谱法,白藜芦醇的检测方法为高效液相色普法。
实施例1 发酵葡萄提取物
制备方法包括以下步骤:
1)将全葡萄原料(包括葡萄藤、葡萄叶、葡萄梗、葡萄皮、葡萄籽等)清洗破碎成浆,加入全葡萄重量0.25‰的酿酒活性干酵母,控制发酵温度25℃发酵14d;
2)将发酵液过滤,得到滤液A;
3)将过滤后的滤渣干燥,粉碎,加入滤渣干重8倍的80%的乙醇,常温条件下提取4h,过滤,得到提取液;
4)将所述滤液A和提取液合并,浓缩至干,得到发酵葡萄粗提物;
5)将所得粗提取物溶于体积比为2∶3的乙醇与乙酸乙酯的混合液,过滤,弃去滤渣,得滤液B;
将滤液B过大孔吸附树脂柱D-101,以乙醇与乙酸乙酯体积比为4∶1的混合液作为洗脱液进行洗脱,收集洗脱液;
6)将所得洗脱液,减压浓缩,将浓缩液在3-5℃条件下静置48析晶,经冷冻干燥,得发酵葡萄提取物。
经检测,本实施例所得发酵葡萄提取物提取率为:32.28mg/g。葡萄多酚含量50%,白藜芦醇含量20%。
实施例2 发酵葡萄提取物
制备方法包括以下步骤:
1)将全葡萄原料(包括葡萄藤、葡萄叶、葡萄梗、葡萄皮、葡萄籽等)清洗破碎成浆,加入全葡萄重量0.2‰的活性干酵母,控制发酵温度25℃发酵7d;
2)将发酵液过滤,得到滤液A;
3)将过滤后的滤渣干燥,粉碎,加入滤渣干重8倍的70%的乙醇,常温条件下提取4h,过滤,得到提取液;
4)将所述滤液A和提取液合并,浓缩至干,得到发酵葡萄粗提物;
5)将所得粗提取物溶于体积比为2∶3的乙醇与乙酸乙酯的混合液,过滤,弃去滤渣,得滤液B;
将滤液B过大孔吸附树脂柱D-101,以乙醇与乙酸乙酯体积比为4∶1的混合液作为洗脱液进行洗脱,收集洗脱液;
6)将所得洗脱液,减压浓缩,将浓缩液在3-5℃条件下静置48析晶,经冷冻干燥,得发酵葡萄提取物。
经检测,本实施例所得发酵葡萄提取物提取率为:25.824mg/g。葡萄多酚含量30%,白藜芦醇含量8%。
实施例3 含实施例1发酵葡萄提取物的片剂
原辅料组成:实施例1发酵葡萄提取物25kg、淀粉4.8kg、硬脂酸镁125g。
制备方法:取实施例1发酵葡萄提取物,粉碎成细粉,过100目筛,以50%的淀粉浆为粘合剂,于一步制粒机内制粒、干燥,加入硬脂酸镁,混匀,压片,包糖衣,即得。
实施例4 含实施例2发酵葡萄提取物的胶囊剂
原辅料组成:实施例2发酵葡萄提取物25kg、硬脂酸镁120g、滑石粉50g。
制备方法:取实施例2发酵葡萄提取物,粉碎成细粉,过120目筛,加入硬脂酸镁和滑石粉,混匀,装胶囊,即得。
实施例5 含实施例1发酵葡萄提取物的口服液
原辅料组成:实施例1发酵葡萄提取物10kg、蔗糖40kg。
制备方法:取实施例1发酵葡萄提取物,加水溶解,加入蔗糖,搅拌使溶解,加水至规定量,煮沸15分钟,待温度降至80℃以下时调节pH值至6.8-7.2,滤过,灌装,灭菌,即得。
实施例6 含实施例2发酵葡萄提取物的颗粒剂
原辅料组成:实施例2发酵葡萄提取物10kg、蔗糖30kg、糊精10kg、硬脂酸镁300g。
制备方法:取实施例2发酵葡萄提取物,粉碎成细粉,过100目筛,加入蔗糖、糊精混匀,制粒,干燥,加入硬脂酸镁总混,分装,即得。
对比例1 验证酵母对提取物中葡萄多酚及白藜芦醇含量的影响
发酵葡萄提取物的制备方法,与实施例1的区别仅在于步骤1)采用面包活性干酵母。
经检测,本实施例所得发酵葡萄提取物提取率为:18.632mg/g。葡萄多酚含量25%,白藜芦醇含量6%。
对比例2 验证萃取溶剂对提取物中葡萄多酚及白藜芦醇含量的影响
发酵葡萄提取物的制备方法,与实施例1的区别仅在于步骤1)采用乙醇为萃取溶剂
经检测,本实施例所得发酵葡萄提取物提取率为:26.232mg/g。葡萄多酚含量14%,白藜芦醇含量3%。
对比例3 验证流速对提取物中葡萄多酚及白藜芦醇含量的影响
发酵葡萄提取物的制备方法,与实施例1的区别仅在于步骤1)采用2ml/min的流速进行洗脱。
经检测,本实验实施例所得发酵葡萄提取物提取率为:23.843mg/g。葡萄多酚含量11%,白藜芦醇含量2%
试验例 抗氧化活性动物实验研究
1、昆明种小鼠40只(日龄30天),雌雄各半,购回后用基础饲料适应喂养10d。
2、随机分成4组,即空白对照组、低剂量组(每天灌喂实施例1制得的发酵葡萄提取物100mg/kg)、中剂量组(每天灌喂实施例1制得的发酵葡萄提取物200mg/kg)、和高剂量组(每天灌喂实施例1制得的发酵葡萄提取物300mg/kg)。各剂量组每天按剂量灌喂发酵葡萄提取物水溶液,空白对照组每天灌喂等量的生理盐水。各组均自由饮水/自由饮食,每周测体重一次,连续饲喂30天。检测小鼠血液抗氧化指标,结果见表1。
表1 发酵葡萄提取物对小鼠血液抗氧化指标的影响
注:发酵葡萄提取物对小鼠血液抗氧化指标的影响(n=10)。与正常对照组比较,#P<0.05,#P<0.01。丙二醛(malondialdehyde,MDA)、超氧化物歧化酶(superoxide dismutase,SOD)、过氧化氢酶(catalase,CAT)、谷胱甘肽过氧化物酶(glutathione peroxidase,GSH-Px)。
3、表1结果表明各剂量组小鼠血浆中MDA含量均极显著低于空白对照组,且灌喂的剂量越大,MDA含量越低,说明发酵葡萄提取物能降低血液中MDA含量。各剂量组小鼠血浆中SOD、CAT、GSH-Px活力均高于空白对照组,且随着剂量的增大,活力增强。与空白对照组比较,低剂量组SOD、CAT活力达显著水平,中、高剂量组SOD、CAT和GSH-Px活力均达极显著水平。
4、实验结果表明发酵葡萄提取物能显著提高抗氧化酶的活力,且功效与剂量呈正相关。
虽然,上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述,但在本发明基础上,可以对之作一些修改或改进,这对本领域技术人员而言是显而易见的。因此,在不偏离本发明精神的基础上所做的这些修改或改进,均属于本发明要求保护的范围。
Claims (10)
1.一种发酵葡萄提取物,含有葡萄多酚和白藜芦醇,其特征在于,葡萄多酚的含量≥30%,白藜芦醇的含量≥8%。
2.根据权利要求1所述的发酵葡萄提取物,其特征在于,所述发酵葡萄提取物中葡萄多酚的含量为30%-50%,白藜芦醇的含量为8%-20%。
3.权利要求1或2所述发酵葡萄提取物的制备方法,其特征在于,包括以下步骤:将葡萄发酵液过滤,得滤液A和滤渣;将滤渣干燥,粉碎,用乙醇提取,得到提取液;将所述滤液A和提取液合并,浓缩至干,得到粗提物;将所得粗提取物溶于体积比为1:1-1:2的乙醇与乙酸乙酯的混合液,过滤,得滤液B;将滤液B过大孔吸附树脂柱,以乙醇与乙酸乙酯体积比为7:3-9:1的洗脱液进行洗脱,收集洗脱液;减压浓缩,静置析晶,冷冻干燥,即得。
4.根据权利要求3所述的制备方法,其特征在于,用于溶解所述粗提物的混合液中乙醇与乙酸乙酯体积比为2∶3;所述洗脱液中乙醇与乙酸乙酯的体积比为4∶1。
5.根据权利要求3或4所述的制备方法,其特征在于,滤液B过大孔吸附树脂柱的流速为1ml/min;所述大孔树脂优选为ADS-F8、ADS-17、HPD-750、D-101、HPD-100、HPD-450或DA-201中的一种。
6.根据权利要求3或4所述的制备方法,其特征在于,所述葡萄发酵液是经酿酒活性干酵母发酵制得的;优选地,所述酿酒活性干酵母的用量为占葡萄原料重量的0.1-0.5‰,进一步优选为0.2-0.25‰。
7.根据权利要求3或4所述的制备方法,其特征在于,所述葡萄发酵液由全葡萄原料制得,包括葡萄藤、葡萄叶、葡萄梗、葡萄皮、葡萄籽。
8.根据权利要求3所述的制备方法,其特征在于,包括以下步骤:
1)将全葡萄原料清洗破碎成浆,加入全葡萄原料重量0.2-0.25‰的酿酒活性干酵母,控制发酵温度10-28℃,发酵7-15d;
2)将发酵液过滤,得到滤液A;
3)将滤渣干燥,粉碎,加入滤渣干重8-15倍的60%-90%的乙醇,提取1-3次,每次1-4h,合并滤液,得到提取液;
4)将所述滤液A和提取液合并,浓缩至干,得到粗提物;
5)将所得粗提取物溶于体积比为1:1-1:2的乙醇与乙酸乙酯的混合液,过滤,得滤液B;将滤液B过大孔吸附树脂柱,以乙醇与乙酸乙酯体积比为7:3-9:1的洗脱液进行洗脱,收集洗脱液;
6)将所得洗脱液,减压浓缩,将浓缩液在3-5℃条件下静置24-72h析晶,冷冻干燥,即得。
9.含权利要求1或2所述发酵葡萄提取物或权利要求3-8任一项所述方法制备的发酵葡萄提取物的制剂,该制剂由所述发酵葡萄提取物和药学上可接受的载体或稀释剂组成。
10.权利要求1或2所述发酵葡萄提取物或权利要求9所述制剂在制备抗氧化功能的保健食品中的应用。
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