CN113475712B - 一种鹿皮胶及制备方法 - Google Patents
一种鹿皮胶及制备方法 Download PDFInfo
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- CN113475712B CN113475712B CN202110871420.7A CN202110871420A CN113475712B CN 113475712 B CN113475712 B CN 113475712B CN 202110871420 A CN202110871420 A CN 202110871420A CN 113475712 B CN113475712 B CN 113475712B
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Abstract
本发明公开了一种鹿皮胶及制备方法,包括如下质量份数的原料:鹿皮36‑43份、鹿骨16‑24份、鹿尾16‑24份、白芍4‑6份、当归2.5‑3.5份、陈皮2.5‑3.5份、山楂2.5‑3.5份、甘草2.5‑3.5份、益母草2.5‑3.5份、黄酒3.5‑4.5份、冰糖3.5‑4.5份;并制备鹿皮胶液、鹿骨鹿尾多肽提取液、中药提取液,再将鹿皮胶液、鹿骨鹿尾多肽提取液、中药提取液混合,混合后加入黄酒和冰糖,加热浓缩后置于模具中,经冷却冷藏得到鹿皮胶。本发明所提供的鹿皮胶选自药食同源品种,不仅发挥药物的有效性,还可以满足食品的安全性;且制备方法简单,对工艺设备要求较低,具有产率好、口感独特、稳定性好、滋补作用显著等优点,受众人群大,具有广阔的市场。
Description
技术领域
本发明涉及保健养生食品技术领域,更具体的是涉及鹿皮、鹿骨、鹿尾综合利用技术领域,具体为一种鹿皮胶及制备方法。
背景技术
鹿皮在我国主要为梅花鹿或者马鹿的皮,它是传统的中药之一,且药用历史悠久。鹿皮是极具经济和研发价值。目前,国内外对鹿皮的研究仅限于鹿皮胶原蛋白的分析,其开发应用处于粗加工阶段,有关鹿皮其他有效成分的提取、精制、深加工技术及应用等方面的相关研究在国内外尚处在空白阶段,并无相关报道。鹿副产品的开发应用,无论是在医药业,还是在食品、保健品及化妆品行业都较少。面对我国目前鹿业产业的发展现状,开发鹿产品深加工技术,尤其是鹿副产品的深加工技术的创新是必然发展趋势。
鹿骨为鹿科动物梅花鹿或马鹿的骨骼。鹿骨骼中营养物质丰富含有大量蛋白质、磷脂质、磷蛋白、多种维生素,还含有多种微量元素,并参与人体多种代谢具有重要的药理作用。但我国多以鹿茸、鹿鞭及鹿胎等原料的销售及深加工为主,有关鹿骨、鹿皮等副产品研究开发较少,特别是鹿皮相关的产品尚未得到很好地开发。骨骼作为鹿的副产物年产量巨大,大部分都未得到充分利用,仅停留于骨制品的初级加工,如骨骼超微粉、肥料等。
进一步,鹿尾为鹿科动物梅花鹿或马鹿的尾巴,是我国名贵的强壮补益中药材。我国传统中医学把鹿尾当做是鹿的阴精聚集之处,因为获取鹿尾比获取鹿茸更加艰难,所以鹿尾在古代就被称之为稀世珍品,几千年来备受人们的推崇。在《四川中药志》中明确记载了鹿尾具有增强体质、提神醒脑、温补壮阳等功效。《随园食单》亦有记载:“文端公品味,以鹿尾为第一”的说法。近年来随着对鹿尾药理性作用、功能性成分研究的不断深入,加之生活水平的日益提高和人们对长寿安康的追求,在我国甚至世界都对滋补作用显著的鹿尾的需求越来越强烈。
由于科学技术高度发达的现代社会的高速发展,人民生活水平的不断提高,人们对营养、保健的要求也大大提高,这就迫切需求寻找、研究开发新的安全有效的治疗药品、保健药品,为增进人类的健康而服务。而随着生活质量的逐步提高,绿色保健型产品也日益为人们所关注,近年来鹿产品更是凭借其卓越的药用保健价值,在医疗保健和食品领域受到广泛重视。
因此,如何开发一种以鹿皮、鹿骨、鹿尾为主要原料的养生品,不仅实现资源的综合利用,提高利用率,且利于人体的消化和吸收,发挥其药用功效,是本领域亟需解决的技术问题。
发明内容
有鉴于此,本发明提供了一种鹿皮胶及其制备方法,主要以鹿皮为主药引,以鹿骨、鹿尾和中药材为辅助;其中鹿皮利用高温高压蒸煮熬制并联用蛋白酶酶解制备成鹿皮胶液,鹿骨和鹿尾采用蛋白酶联用方法制备成鹿骨鹿尾多肽提取液,且对中药提取,再将三者调配,制备成一种具有补气血、提高免疫力和养生保健、美容养颜作用的鹿皮胶。
为了实现上述目的,本发明采用如下技术方案:
一种鹿皮胶,包括以下质量百分含量的原料:
上述技术方案的有益效果:
其中,白芍:味苦、酸,性微寒;归肝、脾经。
当归:味甘、辛,性温;归肝、心、脾经;补血活血,调经止痛,润肠通便。
陈皮:味辛苦,性温;归脾、肺经;具有理气健脾、燥湿化痰的功效。
山楂:酸、甘,微温;归脾、胃、肝经;消食健胃,行气散瘀,化浊降脂。
甘草:味甘,性平;归心、肺、脾、胃经;补脾益气,清热解毒,祛痰止咳,缓急止痛,调和诸药。
益母草:味苦、辛,微寒;归肝、心包、膀胱经;活血调经,利尿消肿,清热解毒。
综上所述,本发明公开的一种鹿皮胶,一方面可以充分发挥鹿皮补气养血,涩虚精,强筋健骨;鹿骨鹿尾补肾壮阳、生精益血、补髓健骨的作用;再辅以当归补气固表,补气生血,甘草清热解毒,补脾益气。山楂陈皮益脾,行气健胃;再加以益母草及白芍活血调经,敛阴止汗,调和药性,诸药联合入药,可达到生精益血、强健骨骼、美容养颜的效果,既补阳又滋阴,共奏补肾益肝、补脾益气之功。另一方面鹿皮胶所选用药材均为药食同源品种,以鹿皮鹿骨和鹿尾为原料,营养均衡,不仅可以发挥药物有效性,还可满足食品安全性。
优选的,所述鹿骨和所述鹿尾的含水量均为12-15%。
优选的,所述鹿皮选自梅花鹿或马鹿的新鲜优质鹿皮。
上述技术方案的有益效果:通过合理控制鹿骨和鹿尾的含水量一方面有利于后续对原料的处理,同时有效提高酶解的效率。
本发明还公开了一种鹿皮胶的制备方法,具体包括如下步骤:
1)根据所述的鹿皮胶称取各个原料,备用;
2)鹿皮胶液的制备:采用高温高压蒸煮+蛋白酶联用酶解的方法对鹿皮进行预处理得到鹿皮提取液,再进行加热浓缩得到鹿皮胶液;
3)鹿骨鹿尾多肽提取液的制备:
31)分别对鹿骨和鹿尾进行预处理,并均破碎至20-30mm;再经烘干、粉碎得到鹿骨粉和鹿尾粉;
32)分别用去离子水对鹿骨粉和鹿尾粉进行浸泡、研磨得到鹿骨粉悬浮液、鹿尾粉悬浮液,然后将等量的鹿骨粉悬浮液和鹿尾粉悬浮液混合后加入去离子水,进行回流提取,经过滤得到鹿骨鹿尾蛋白粗提取液;
33)采用胰蛋白酶-无花果蛋白酶联用对鹿骨鹿尾蛋白粗提取液进行酶解,经离心、过滤得鹿骨鹿尾多肽提取液;
4)中药提取液的制备:将白芍、当归、陈皮、山楂、甘草及益母草混合后烘干、粉碎、过筛得中药粉末,将中药粉末浸泡于去离子水中超声提取多次,经离心得中药提取液;
5)将鹿皮胶液、鹿骨鹿尾多肽提取液、中药提取液混合,混合后加入黄酒和冰糖,加热浓缩后置于模具中,经冷却冷藏得到鹿皮胶。
采用上述技术方案的有益效果:本发明提供了一种制备鹿皮胶的方法,采用超声提取法处理中药材,破碎其中组织,使溶出物增加,营养成分利用更充分。采用胰蛋白酶酶-无花果蛋白酶联用酶解技术,酶解更完全,效果更佳;同时对鹿骨和鹿尾进行粉碎,进一步提高酶解的程度,使其更加完全。最终得到了一种营养丰富,增强免疫、缓解体力疲劳,美容养颜滋补养生的鹿皮胶。
本发明的制备方法简单,对工艺设备要求比较低。具有产率良好、口感独特、稳定性好、滋补作用显著等优点。受众人群大,具有广阔的市场。
本发明所制备的鹿皮胶所选用药材均为药食同源品种,以鹿皮鹿骨和鹿尾为原料,营养均衡,不仅可以发挥药物有效性,还可满足食品安全性。
本发明将调配出鹿皮、鹿骨和鹿尾最好的有效成分利用率并发挥出最大的药用效果,且口感较好。
本发明为保健食品,其药物组合物用药剂量小,疗效好,方便使用,价格低廉,服用简单,日常服用具有养生保健之效,适宜各类人群。
本发明的鹿骨鹿尾多肽提取液制备中,采用胰蛋白酶-无花果蛋白酶联用酶解。胰蛋白酶是一种蛋白水解酶,存在于人和动物的消化道中,也是动物胰脏内的主要蛋白酶。它能够催化蛋白质的特定肽键水解,不同的蛋白酶可以作用在不同氨基酸相连组成的肽键。无花果蛋白酶是一种含巯基肽链内切酶,具有蛋白酶和酯酶的活性,可改变游离氨基和蛋白质表面疏水性,有较广泛的特异性,对动植物蛋白、多肽、酯、酰胺等有较强的水解能力。二者联用,可有效将鹿骨鹿尾多肽提取液中大分子蛋白质酶解成游离氨基酸和小分子多肽易于人体的消化与吸收。
进一步,本发明分别制作了三种提取液,鹿皮提取液,鹿骨鹿尾多肽提取液以及中药材提取液,此方法制备简单,可根据原料的特性和差异性采用不同的制备工艺制备,发挥其最好的药用功效。
同时,通过中药材的加入,有效缓和了鹿皮、鹿骨和鹿尾所带来的口感腥腻的味道,使得制备的鹿皮胶口感醇厚香甜,老少皆宜;所加的中药材提取液与鹿皮、鹿骨和鹿尾,起到协同增效的积极作用,有效增强了鹿皮胶的保健养生功效,并且提升了鹿皮胶的口感,使得人们短时间服用就能起到保健的功效。
更进一步,黄酒的加入不仅可以去除鹿皮的腥味,保质防腐,还可以增加鹿皮胶的功效,促进药效挥发,使鹿皮胶易于消化吸收,并且黄酒还具有通血脉,补气养血的作用。
优选的,步骤2)所述的鹿皮胶液的制备具体为:
21)去除鹿皮表面杂质污垢,且冲洗去除血渍和污渍,再将鹿皮切割成(5-6)cm*(5-6)cm的鹿皮块;
22)将鹿皮块置于沸水中烫煮至卷缩状,并除去表面残肉和脂肪,沥干后绞碎并置于密闭的高温高压锅内,加水提取,过滤得鹿皮提取液;
23)采用胰蛋白酶-无花果蛋白酶联用对鹿皮提取液进行酶解,酶解完毕后加热浓缩得鹿皮胶液。
优选的,步骤21)去除鹿皮表面杂质污垢是采用浸泡的方式实现的,然后在流水中冲洗9-12min,洗净毛皮,并除去鹿皮中的血渍和污渍。
进一步,步骤22)所述鹿皮块置于沸水中烫煮需要不断的上下翻动至卷缩状,且用刮刀刮去表面残肉和脂肪。
优选的,步骤22)所述高温高压的压力为0.09-0.15MPa、温度为100-120℃,提取时间4-6h;
所述加水量为所述鹿皮质量的4-6倍;
采用上述技术方案的有益效果:鹿皮采用高温高压蒸煮联用蛋白酶酶解的方式,可以有效获取鹿皮中的有效成分胶原蛋白肽;但是由于鹿皮表面粗糙,皮质较厚而且韧性较强,在加工过程中存在组织不易捣碎、酶解效率低等问题,因此,采用高温高压蒸煮处理的鹿皮不仅可以去除掉部分死皮、色素等杂质,更重要的是可以软化组织,使鹿皮膨胀,提高后续制备胶原蛋白肽的利用率,再通过蛋白酶的联用,可以将鹿皮中的有效成分大分子蛋白,酶解成小分子胶原蛋白肽,更有利于后续人体的吸收和利用。
进一步,步骤22)所述提取完毕后用纱布过滤,同时对滤液进行搅拌至均匀,搅拌速度为200r/min,静置3h后再用八层纱布过滤得鹿皮提取液。
优选的,步骤23)所述胰蛋白酶的加入量为鹿皮质量的0.6-0.8%,酶解温度35-40℃,酶解时间55-65min。
所述无花果蛋白酶的加入量为鹿皮质量的0.6-0.8%,酶解温度50-55℃,酶解时间55-65min。
优选的,步骤31)所述的预处理包括:对所述鹿骨除净肌肉,筋膜和骨髓,再烘干至含水量至12%;
所述鹿尾祛毛洗净后,烘干至含水量至12%;
所述破碎采用锤片式破碎机破碎。
优选的,步骤31)所述烘干的温度60-70℃,烘干时间120-150min;
所述粉碎后过60目筛;
步骤32)所述浸泡时间3-4h,研磨时间8-10min,且所述鹿骨粉悬浮液和鹿尾粉悬浮液混合后与去离子水的料液比为1g:10ml,提取温度90℃,提取时间3h。
进一步,所述鹿骨粉、所述鹿尾粉用胶体磨循环研磨,研磨速度2000r/min。
优选的,步骤33)所述胰蛋白酶-无花果蛋白酶联用其中胰蛋白酶的添加量为鹿骨粉和鹿尾粉总质量的0.6-0.8%,酶解时间35-40℃,酶解温度55-65min,酶解完毕后沸水浴灭酶10min;
所述无花果蛋白酶的添加量为鹿骨粉和鹿尾粉总质量的0.6-0.8%,酶解时间50-55℃,酶解温度55-65min,酶解完毕后沸水浴灭酶10min。
更进一步,步骤33)所述离心速度为3500-4000r/min,离心时间为9-12min。
上述技术方案的有益效果:利用胰蛋白酶-无花果蛋白酶联用酶解法提取鹿骨、鹿尾多肽的方法,提高多肽等有效成分和活性成分的提取率,最大限度的保留了生物活性物质,使其生物利用率大大提高;进一步提高人体的吸收率,减少消化器官的负担。
同时,胰蛋白酶是一种蛋白水解酶,存在于人和动物的消化道中,也是动物胰脏内的主要蛋白酶,它能够催化蛋白质的特定肽键水解,不同的蛋白酶可以作用在不同氨基酸相连接组成的肽键。
无花果蛋白酶是一种含巯基肽链内切酶,具有蛋白酶和酯酶的活性,可改变游离氨基和蛋白质表面的疏水性,有较广泛的特异性,对动植物蛋白,多肽、酯、酰胺等有较强的水解能力;因此,二者联用,可有效将鹿骨鹿尾多肽提取液中大分子蛋白质和游离氨基酸酶解成小分子多肽,易于人体吸收和消化。
优选的,步骤4)所述白芍、当归、陈皮、山楂、甘草及益母草混合之前先清洗干净;所述烘干温度50℃,烘干时间60min,所述过60目筛。
更进一步,所述中药粉末与去离子水的体积比为1:(9-11),所述提取多次,至少提取两次,且所述超声提取功率为100-120w,所述提取温度50-70℃,所述提取时间60-80min;
且步骤4)所述离心速度为3500-4000r/min,离心时间为9-12min。
采用上述技术方案的有益效果:通过中药的加入,有效缓和了鹿皮、鹿骨和鹿尾所带来的口感腥腻的味道,同时使制备的鹿皮胶口感醇厚香甜,老少皆宜;且中药提取液与鹿皮、鹿骨和鹿尾,起到了协同增效的积极作用,有效增强了鹿皮胶的保健养生的功效,提升了鹿皮胶的口感,实现保健养生的目的。
优选的,步骤5)所述冷藏温度0-4℃;且步骤2)和步骤5)所述加热浓缩至用玻璃棒挑起成线即可。
更进一步,步骤5)所述的加热浓缩的温度90-95℃,所述冷却温度2-4℃。
综上所述,与现有技术相比,本发明至少具有如下技术优点:
(1)采用高温高压蒸煮且联用蛋白酶酶解的方法熬制鹿皮,可有效提取鹿皮中的有效成分;
(2)采用胰蛋白酶-无花果蛋白酶联用酶解技术制备鹿骨和鹿尾多肽悬浮液的方法,提高了鹿骨和鹿尾多肽等活性成分的提取率;
(3)将中药材泡制于去离子水中,采用超声提取法,可以加速有效成分的溶出;本发明以鹿皮胶液为君,以鹿骨、鹿尾多肽提取液和中药提取液为臣,制备成鹿皮胶,不仅提高人体的吸收率,而且还具有保健的功能;
(4)本发明所提供的鹿皮胶其原料选自药食同源品种,不仅发挥药物的有效性,还可以满足食品的安全性;且制备方法简单,对工艺设备要求较低,具有产率好、口感独特、稳定性好、滋补作用显著等优点,受众人群大,具有广阔的市场;
(5)本发明制备的鹿皮胶为保健养生食品,其中药组合物用药剂量小,疗效好、方便使用、价格低廉、服用简单,日常服用具有养生保健之功效,适宜各类人群。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据提供的附图获得其他的附图。
图1附图为本发明实施例11的羟基自由基清除检测示意图;
具体实施方式
下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
本发明实施例1-5及对比例1-2均提供了一种鹿皮胶,并按照表1包括如下质量(g)的原料。
表1
本发明实施例6还提供了一种鹿皮胶的制备方法,其具体包括:
1)按照配比称取鹿皮、鹿骨、鹿尾、白芍、当归、陈皮、山楂、甘草、益母草、黄酒和冰糖;
2)鹿皮胶液的制备:采用高温高压蒸煮+蛋白酶联用酶解的方法对鹿皮进行预处理得到鹿皮提取液,再进行加热浓缩得到鹿皮胶液;
3)鹿骨鹿尾多肽提取液的制备:
32)分别对鹿骨和鹿尾进行预处理,并均破碎至20-30mm;再经烘干、粉碎得到鹿骨粉和鹿尾粉;
32)分别用去离子水对鹿骨粉和鹿尾粉进行浸泡、研磨得到鹿骨粉悬浮液、鹿尾粉悬浮液,然后将等量的鹿骨粉悬浮液和鹿尾粉悬浮液混合后加入去离子水,进行回流提取,经过滤得到鹿骨鹿尾蛋白粗提取液;
33)采用胰蛋白酶-无花果蛋白酶联用对鹿骨鹿尾蛋白粗提取液进行酶解,经离心、过滤得鹿骨鹿尾多肽提取液;
4)中药提取液的制备:将白芍、当归、陈皮、山楂、甘草及益母草混合后烘干、粉碎、过筛得中药粉末,将中药粉末浸泡于去离子水中超声提取多次,经离心得中药提取液;
5)将鹿皮胶液、鹿骨鹿尾多肽提取液、中药提取液混合,混合后加入黄酒和冰糖,加热浓缩后置于模具中,经冷却冷藏得到鹿皮胶。
为了进一步优化上述技术方案,步骤2)的鹿皮胶液的制备具体为:
21)去除鹿皮表面杂质污垢,且冲洗去除血渍和污渍,再将鹿皮切割成(5-6)cm*(5-6)cm的鹿皮块;
22)将鹿皮块置于沸水中烫煮至卷缩状,并除去表面残肉和脂肪,沥干后绞碎并置于密闭的高温高压锅内,加水提取,过滤得鹿皮提取液;
23)采用胰蛋白酶-无花果蛋白酶联用对鹿皮提取液进行酶解,酶解完毕后加热浓缩得鹿皮胶液。
为了进一步优化上述技术方案,步骤21)去除鹿皮表面杂质污垢是采用浸泡的方式实现的,然后在流水中冲洗9-12min,洗净毛皮,并除去鹿皮中的血渍和污渍。
为了进一步优化上述技术方案,步骤22)鹿皮块置于沸水中烫煮需要不断的上下翻动至卷缩状,且用刮刀刮去表面残肉和脂肪。
为了进一步优化上述技术方案,步骤22)高温高压的压力为0.09-0.15MPa、温度为100-120℃,提取时间4-6h;
加水量为所述鹿皮质量的4-6倍。
为了进一步优化上述技术方案,步骤22)提取完毕后用纱布过滤,同时对滤液进行搅拌至均匀,搅拌速度为200r/min,静置3h后再用八层纱布过滤得鹿皮提取液。
为了进一步优化上述技术方案,步骤23)胰蛋白酶的加入量为鹿皮质量的0.6-0.8%,酶解温度35-40℃,酶解时间55-65min。
无花果蛋白酶的加入量为鹿皮质量的0.6-0.8%,酶解温度50-55℃,酶解时间55-65min。
为了进一步优化上述技术方案,步骤31)的预处理包括:对所述鹿骨除净肌肉,筋膜和骨髓,再烘干至含水量至12%;
鹿尾祛毛洗净后,烘干至含水量至12%;
破碎采用锤片式破碎机破碎。
为了进一步优化上述技术方案,步骤31)烘干的温度60-70℃,烘干时间120-150min;
粉碎后过60目筛;
步骤32)浸泡时间3-4h,研磨时间8-10min,且鹿骨粉悬浮液和鹿尾粉悬浮液混合后与去离子水的料液比为1g:10ml,提取温度90℃,提取时间3h。
为了进一步优化上述技术方案,鹿骨粉、鹿尾粉均用胶体磨循环研磨,研磨速度为2000r/min。
为了进一步优化上述技术方案,步骤33)胰蛋白酶-无花果蛋白酶联用其中胰蛋白酶的添加量为鹿骨粉和鹿尾粉总质量的0.6-0.8%,酶解时间35-40℃,酶解温度55-65min,酶解完毕后沸水浴灭酶10min;
无花果蛋白酶的添加量为鹿骨粉和鹿尾粉总质量的0.6-0.8%,酶解时间50-55℃,酶解温度55-65min,酶解完毕后沸水浴灭酶10min。
为了进一步优化上述技术方案,步骤33)离心速度为3500-4000r/min,离心时间为9-12min。
为了进一步优化上述技术方案,步骤1)白芍、当归、陈皮、山楂、甘草及益母草混合之前先清洗干净;所述烘干温度50℃,烘干时间60min,所述过60目筛。
为了进一步优化上述技术方案,中药粉末与去离子水的体积比为1:(9-11),提取多次至少提取两次,且超声提取功率为100-120w,提取温度50-70℃,提取时间60-80min;
且步骤4)离心速度为3500-4000r/min,离心时间为9-12min。
为了进一步优化上述技术方案,步骤5)冷藏温度0-4℃;且步骤2)和步骤5)加热浓缩至用玻璃棒挑起成线即可。
为了进一步优化上述技术方案,步骤5)的加热浓缩的温度90-95℃,所述冷却温度2-4℃。
实施例7
依据实施例6对实施例1-5及对比例1-2即表1所示鹿皮胶原料制备相应的鹿皮胶的对应参数如表2所示。
表2
实施例8
根据实施例7制备实施例1-5及对比例1-2的鹿皮胶,并对制备的鹿皮胶进行色泽外观等方面的观察,结果如表3。
表3
实施例9
本实施例以实施例1制备的鹿皮胶进行实验。选取140名志愿者分为平均7组,每组20人进行调研。志愿者标准:志愿者身体处于亚健康状态,出现身体易疲劳,精神不振,免疫力低下,面色暗淡,无光泽,手脚乏力、缺钙,记录改善人数,计算改善率。其中组1至组5为实施例1中所述鹿皮胶,组6与组7分别为对比例1和对比例2中所述鹿皮胶。志愿者每天服用两次,每次服用10g,连续服用三个月;结果见表4所示。
表4
实施例10
免疫活性测定
步骤一:脾淋巴细胞悬液制备
取SPF级昆明种小鼠,脱颈处死,无菌摘取脾脏,过200目网筛,用Hank’s液洗涤2-3次,1500r/min离心10min,加入红细胞裂解液破红细胞3-5min;1500r/min离心7min,弃上清;再用RPMIl640培养液洗涤。台盼蓝染色后计数,活细胞数应大于95%,用含10%小牛血清的RPMI1640培养液调整细胞浓度为5×106个/mL,备用。
步骤二:小鼠脾淋巴细胞增殖实验
采用MTT法进行检测。调整其浓度为1.0×106个/ml备用。按照实施例1制备鹿皮胶液、鹿骨鹿尾多肽提取液及鹿皮胶;且将实施例1制备的鹿皮胶液和鹿骨鹿尾多肽提取液进行混合得到鹿皮胶液+鹿骨鹿尾多肽提取液,依据表5中分组进行样液干预。取上述步骤制备得到的脾脏淋巴细胞加入96孔培养板,调节细胞浓度为1.0×105个/mL,每孔100μL细胞,并加1640培养液调整药物浓度;置37℃、5%CO2培养箱内孵育44h,加入20μL MTT(5g/L),继续培养4h,平板离心机离心2000r/min 10min,弃上清液,每孔加入150μL DMSO,37℃振荡10min,于酶标仪492nm处测定OD值。
表5不同浓度各母液对脾淋巴细胞培养结果
由表5可见,不同浓度的鹿皮胶液、鹿骨鹿尾多肽提取液、鹿皮胶液+鹿骨鹿尾多肽提取液,实施例1对小鼠脾脏细胞均有一定的刺激增殖作用,其增殖能力与样液浓度正相关。其中,鹿皮胶液+鹿骨鹿尾多肽提取液均高于其他各组,实施例1鹿皮胶OD值>鹿皮胶液+鹿骨鹿尾多肽提取液OD值>鹿骨鹿尾多肽提取液OD值>鹿皮胶液OD值。显示实施例1以及鹿皮胶液+鹿骨鹿尾多肽提取液与鹿皮胶液、鹿骨鹿尾多肽提取液相比具有较好促进脾脏细胞增殖的作用。淋巴细胞是介导细胞免疫的一种重要反应,可以抵抗细胞内微生物感染,其数量和功能直接对细胞免疫具有决定性的作用,淋巴细胞增殖是反映细胞免疫最直接的指标。
实施例11
羟基自由基清除能力检测
原理:羟自由基(·OH)是活性氧中的一种,而且其具有很强的氧化能力。作为活性氧的一类,羟自由基是机体内反应性和破坏性极强的自由基,与活细胞许多成分反应,直接损伤各种生物大分子和生物膜,引发各种疾病并加快机体衰老,对机体产生危害。因此研究生物体内自由基的作用机制,必须对·OH进行检测。在反应中的过氧化氢和亚铁离子混合发生反应,能够生成具有很高反应活性的·OH。水杨酸能够与·OH高效结合,并且生成有色物质。倘若加入具有清除自由基的物质之后,这种物质就会与水杨酸产生强烈的竞争,从而造成羟自由基与水杨酸的反应量的急剧减少,结果就是有色物质的生成急剧减少,在分光光度计上检测其吸光度值低,说明反应生成的有色物质越少,清除率反而越高。
具体包括:
(1)检测样品:Vc(以蒸馏水为溶剂)、实施例1、对比例1、对比例2中制备的鹿皮胶。
(2)检测方法:取9mmol/L水杨酸钠-乙醇溶液2mL,加入2mL 9mmol/L硫酸亚铁溶液,分别加入2mL不同浓度(0.1、0.2、0.3、0.4、0.5mg/mL)的样品溶液,最后加入2mL8.8mmol/L双氧水。37℃水浴反应30min,在510nm下测定吸光度(Ax)。同时测定用蒸馏水代替硫酸亚铁溶液的吸光度(Ay),蒸馏水代替样品溶液作为空白对照的吸光度(A0)。用下列公式计算清除率(E%)。
清除率=[1-(Ax-Ay)/A0]×100%,式中:
Ax:加入硫酸亚铁后不同浓度鹿皮胶溶液或Vc样品的吸光度;
Ay:蒸馏水代替硫酸亚铁溶液时的吸光度;
A0:蒸馏水空白对照溶液的吸光度。
检测结果见图1所示。
实施例12
参照《中国药典》2015年版阿胶含量测定,本实施例研究建立了鹿皮胶和驴皮胶在L-羟脯氨酸、甘氨酸、丙氨酸、L-脯氨酸的HPLC含量测定,以说明鹿皮胶的有效性。
对照品溶液的制备:精密称取L-羟脯氨酸对照品、甘氨酸对照品、丙氨酸对照品、L-脯氨酸对照品,加0.1mol·L-1盐酸溶液使溶解并定容至50mL。精密量取3mL置10mL量瓶中,加0.1mol·L-1盐酸溶液稀释至刻度,摇匀,滤过,即得混合对照品溶液,每1mL含L-羟脯氨酸80μg、甘氨酸160μg、丙氨酸70μg、L-脯氨酸120μg。
鹿皮胶供试品溶液的制备:取鹿皮胶粗粉0.25g,精密称定,置25ml量瓶中,加0.1mol/L盐酸溶液20ml,加0.1mol/L盐酸溶液至刻度,摇匀。精密量取2ml,150℃水解1小时,放冷,移至蒸发皿中,用水10ml分次洗涤,洗液并入蒸发皿中,蒸干,残渣加0.1mol/L盐酸溶液溶解,移至25ml量瓶中,加0.1mol/L盐酸溶液至刻度,摇匀,即得;
注意:鹿皮胶粗粉是本实施例1制备的鹿皮胶粉碎后过60目筛得到的;
并按照鹿皮胶供试品溶液的制备方法配置驴皮胶供试品溶液;
且驴皮胶的制备方法包括如下步骤:
去除驴皮表面杂质污垢,且冲洗去除血渍和污渍,再将驴皮切割成(5-6)cm*(5-6)cm的驴皮块;将驴皮块置于沸水中烫煮至卷缩状,并除去表面残肉和脂肪,沥干后绞碎并置于密闭的高温高压锅内,加水提取,过滤得驴皮提取液;再将驴皮提取液加热浓缩至用玻璃棒挑起成线即可;最后进行冷藏温度,冷藏温度0-4℃,制得驴皮胶。
其中,高温高压的压力为0.09-0.15MPa、温度为100-120℃,提取时间4-6h;
加水量为驴皮质量的4-6倍。
色谱条件及系统适用性试验:Waters Sunfire C18色谱柱(4.6mm×250mm,5μm);以乙腈-0.1mol·L-1醋酸钠溶液(用36%乙酸调节pH值至6.5)(7∶93)为流动相A,以乙腈-水(4∶1)为流动相B,梯度洗脱(0~13min,100%A→93%A;13~17.9min,93%A→88%A;17.9~29min,88%A→85%A;29~39min,85%A→66%A;39~45min,66%A→0%A);流速1.0mL·min-1;测定波长254nm;柱温43℃;分别吸取对照品和供试品衍生化溶液,进样量5μL。在上述条件下,4种氨基酸的色谱峰与相邻色谱峰的分离度均大于1.5,理论板数均大于6000。
表6鹿皮胶与驴皮胶4种氨基酸含量比较(%)
本实施例中检测所得驴皮胶及鹿皮胶中氨基酸含量均达到《中国药典》2020年版规定水平,鹿皮胶和驴皮阿胶含量相差不大,但各氨基酸含量均略高于驴皮阿胶。证明鹿皮胶同驴皮阿胶一样具有产品深加工的潜力。
实施例13
鹿骨鹿尾蛋白粗提液蛋白酶解前后多肽含量的测定
多肽是指分子结构介于氨基酸和蛋白质之间的一类化合物,且氨基酸是组成多肽和蛋白质的基本基团。
多肽不仅仅易被消化吸收,而且某些具有活性的多肽还同时具有防病、治病、调节人体生理机能的功效,而这些功效是前体蛋白质所不具有的。
多肽含量测定方法:将样品离心后的上清液中的蛋白质,只包括游离氨基酸和多肽,测定酸溶蛋白质的总氮,扣除其中的游离氨基酸,即为多肽的含量。
由上述可知:加入单宁酸可以将大分子蛋白质与小分子多肽分离,且速度较快,节省测定时间,提高效率。
本实施例采用单宁酸作为大分子蛋白质的沉淀剂,结合茚三酮显色法和凯氏定氮仪测定酶解前后的鹿骨鹿尾蛋白粗提液多肽的得率。
1)样品处理:
抽取酶解前与酶解后的鹿骨鹿尾蛋白粗提液样品各3mL,经离心机4000r/min离心10min,吸取上清液1.0mL注入比色管中,再加入质量分数为15%的单宁酸3mL,室温静置沉淀10min,用离心机4000r/min离心30min。
2)上清液中游离氨基酸的含量:
分别准确量取步骤1)两种样品上清液1.0mL,并均置于25mL比色管中,加入2.0mL茚三酮溶液(0.085g/ml),2.0mL抗坏血酸溶液(0.02g/ml),5.0mL乙酸-乙酸钠缓冲溶液(2mol/L),加水至25mL,摇匀。
在分别置于沸水浴中加热20min进行显色,取下冷却至室温,定容至25ml,在568nm波长下比色,在标准曲线上查出相应的游离氨基酸含量A。
3)上清液中多肽含量的测定:
分别准确移取上述步骤中的两种样品上清液2.0mL,并均加入消化管中,之后依次加入0.5g混合催化剂、98%的浓硫酸10mL,打开消化炉,待消化炉的温度升到421℃时计时开始,让其消化1.0h后,关闭消化炉,冷却至室温;
其中,混合催化剂包括:0.3g硫酸铜、0.1g氧化汞、0.1g硒粉。
将消化管放入凯氏定氮仪,关上安全门,等待仪器蒸馏,蒸馏结束,取出消化管和收集氨气的锥形瓶,之后用体积分数0.01%的盐酸标准液滴定,锥形瓶里的液体溶液由蓝绿色变为微红色时为滴定终点;记录盐酸标准溶液消耗的体积,同时测定空白值。
4)上清液中多肽含量的计算
式中:W为蛋白质的质量浓度g/100mL;
C为盐酸标准液的浓度mol/L;
V1为空白滴定消耗的标准盐酸的体积mL;
V2为样品消耗滴定消耗的标准盐酸的体积mL;
2为取样品的体积2mL;
M为氮的摩尔质量14.01g/mol;
6.25为蛋白质的系数。
T=W-A
式中:T为多肽的含量;A为游离氨基酸的含量。
同一样品测定3次,结果如表7所示。
表7鹿骨鹿尾蛋白粗提液酶解前后的多肽含量
由表7可知,经胰蛋白酶-无花果蛋白酶酶解后的鹿骨鹿尾多肽提取液多肽含量较酶解前有明显提高。
总之,本发明实施例公开了一种鹿皮胶其制备方法,采用鹿皮胶液,鹿骨鹿尾多肽提取液,中药提取液,将提取的各组溶液进行科学合理的搭配而制成,经加工成受消费者青睐的一种营养丰富,增强免疫、缓解体力疲劳、美容养颜的食品鹿皮胶,并且该鹿皮胶针对肝肾虚损所致的少气无力,气短,面黄肌瘦,精神不振,腰腿软弱等症状人群有显著药效;鹿皮胶不仅具有服用方便、食用安全等优点,其应用的药材也具有既是食品又兼具药品的天然食物,除疗效确切的功能外兼具养生保健之功效,在保健身体、改善体质虚弱人群中具有较大的市场。
本说明书中各个实施例采用递进的方式描述,每个实施例重点说明的都是与其他实施例的不同之处,各个实施例之间相同相似部分互相参见即可。
对所公开的实施例的上述说明,使本领域专业技术人员能够实现或使用本发明。对这些实施例的多种修改对本领域的专业技术人员来说将是显而易见的,本文中所定义的一般原理可以在不脱离本发明的精神或范围的情况下,在其它实施例中实现。因此,本发明将不会被限制于本文所示的这些实施例,而是要符合与本文所公开的原理和新颖特点相一致的最宽的范围。
Claims (2)
1.一种鹿皮胶,其特征在于,由以下质量的原料组成:
鹿皮 40g
鹿骨 20g
鹿尾 20g
白芍 5g
当归 3g
陈皮 3g
山楂 3g
甘草 3g
益母草 3g
黄酒 4g
冰糖 4g;
具体包括如下步骤:
1)称取各个原料,备用;
2)鹿皮胶液的制备:采用高温高压蒸煮和蛋白酶联用酶解的方法对鹿皮进行预处理得到鹿皮提取液,再进行加热浓缩至用玻璃棒挑起成线即可,得到鹿皮胶液,具体为:
2.1)采用浸泡方式去除鹿皮表面杂质污垢,在流水中冲洗9-12min,洗净毛皮,去除血渍和污渍,再将鹿皮切割成5-6cm*5-6cm的鹿皮块;
2.2)将鹿皮块置于沸水中烫煮至卷缩状,并除去表面残肉和脂肪,沥干后绞碎并置于密闭的高温高压锅内,加水提取,过滤得鹿皮提取液;
其中,所述压力为0.12MPa、温度为110℃,提取时间5h;高温高压锅内水的体积为100ml;
2.3)采用胰蛋白酶-无花果蛋白酶联用对鹿皮提取液进行酶解,酶解完毕后加热浓缩得鹿皮胶液;
其中,所述胰蛋白酶的加入量为0.25g,酶解温度37℃,酶解时间60min,沸水灭酶时间为10min;
所述无花果蛋白酶的加入量为0.25g,酶解温度52℃,酶解时间60min,沸水灭酶时间为10min;
3)鹿骨鹿尾多肽提取液的制备:
3.1)分别对鹿骨和鹿尾进行预处理,对所述鹿骨除净肌肉,筋膜和骨髓,再烘干至含水量至12%,所述鹿尾祛毛洗净后,烘干至含水量至12%;并均破碎至20-30mm,所述破碎采用锤片式破碎机破碎;再经烘干、粉碎得到鹿骨粉和鹿尾粉;
其中,烘干的温度60-70℃,烘干时间120-150min;粉碎后过60目筛;
3.2)分别用去离子水对鹿骨粉和鹿尾粉进行浸泡、研磨得到鹿骨粉悬浮液、鹿尾粉悬浮液,然后将等量的鹿骨粉悬浮液和鹿尾粉悬浮液混合后加入去离子水,进行回流提取,经过滤得到鹿骨鹿尾蛋白粗提取液;
其中,所述浸泡时间4h,研磨时间10min,且所述鹿骨粉悬浮液和鹿尾粉悬浮液混合后与去离子水的料液比为1g:10ml,提取温度90℃,提取时间3h;
3.3)采用胰蛋白酶-无花果蛋白酶联用对鹿骨鹿尾蛋白粗提取液进行酶解,经离心、过滤得鹿骨鹿尾多肽提取液;
其中,离心速率为3500r/min,离心时间为10min;
所述胰蛋白酶的添加量为0.28g,酶解时间37℃,酶解温度60min,沸水灭酶时间为10min;
所述无花果蛋白酶的添加量为0.28g,酶解时间52℃,酶解温度60min,沸水灭酶时间为10min;
4)中药提取液的制备:将白芍、当归、陈皮、山楂、甘草及益母草清洗干净后混合,并在50℃烘干60min、粉碎过60目筛得中药粉末,将中药粉末浸泡于去离子水中超声提取2次,经离心得中药提取液;
其中,提取温度为70℃,提取功率110w,提取时间60min,离心速率为3500r/min,离心时间为10min;
5)将鹿皮胶液、鹿骨鹿尾多肽提取液、中药提取液混合,混合后加入黄酒和冰糖,加热浓缩至用玻璃棒挑起成线即可,再置于模具中,经冷却冷藏得到鹿皮胶;
其中,所述加热浓缩的温度90-95℃,所述冷却温度2-4℃,所述冷藏温度4℃。
2.根据权利要求1所述的一种鹿皮胶,其特征在于,所述鹿皮选自梅花鹿或马鹿的新鲜优质鹿皮。
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