CN110801024A - 降低血糖血脂及糖化血红蛋白的多糖复合多肽及制备方法 - Google Patents
降低血糖血脂及糖化血红蛋白的多糖复合多肽及制备方法 Download PDFInfo
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Abstract
本发明公开了一种降低血糖血脂及糖化血红蛋白的多糖复合多肽及制备方法,按重量份计,其包括:燕麦膳食纤维粉20‑25粉、魔芋精粉10‑15份、玉米须10‑15份、苦瓜肽粉20‑30份、大豆多肽10‑12份、桑叶提取物5‑10份、栀子果油5‑10份,可可粉5‑10份、L‑***糖5‑10份、茯苓提取物3‑5份、山楂提取物5‑10份、营养酵母1‑2份、胰酶2‑5份、木糖醇5‑8份。其通过特殊的苦瓜肽提取工艺来提高苦瓜肽活性成分的含量,且通过与其他天然植物组分的配合使用来达到更好的降血糖、糖化血红蛋白的效果。
Description
技术领域
本发明涉及营养功能性食品与生物发酵领域。更具体地说,本发明涉及一种降低血糖血脂及糖化血红蛋白的多糖复合多肽及制备方法。
背景技术
糖尿病、高脂血症和肥胖症是近年来发病率较高的几种疾病,随着人们生活水平的提高,其发病率成上升趋势,且不但老年人易得上述疾病,年轻人发病率也在增长。虽然近年来预防上述疾病的中、西药以及保健食品纷纷面市,但仍有许多疑难问题需要人们去解决,不断开发防治糖尿病,高脂血症和肥胖症的新型药物、保健食品仍是摆在人们面前的重大课题。
血糖和糖化血红蛋白均是衡量糖尿病,高脂血症和肥胖症症状的重要指标。其中,血糖是从食物中的碳水化合物分解而来的血液中的单糖,通常仅指葡萄糖,血糖测试结果反映的是即刻的血糖水平。糖化血红蛋白(HbA1c)是血液葡萄糖通过非酶作用,经细胞膜与红细胞内血红蛋白-链颉氨酸结合形成的产物,其合成速率与红细胞所处环境中糖的浓度成正比,是人体血液中红细胞内的血红蛋白与血糖结合的产物。血糖和血红蛋白的结合生成糖化血红蛋白是不可逆反应,并与血糖浓度成正比,且保持120天左右。所以糖化血红蛋白较血糖测定更有临床意义,被誉为糖尿病病情监测的“金标准”,在临床检测中进行日常检测作为血糖控制的指标。
因此,如何提供一种能更有效调控血糖的降低血糖及糖化血红蛋白的食品、药品显得尤为重要。
发明内容
为解决上述技术问题,本发明提供了一种降低血糖血脂及糖化血红蛋白的多糖复合多肽及制备方法,其通过特殊的苦瓜肽提取工艺来提高苦瓜肽活性成分的含量,且通过与其他天然植物组分的配合使用来达到更好的降血糖、糖化血红蛋白的效果。
为了实现根据本发明的这些目的和其它优点,提供了一种降低血糖血脂及糖化血红蛋白的多糖复合多肽,按重量份计,其包括:燕麦膳食纤维粉20-25份、魔芋精粉10-15份、玉米须10-15份、苦瓜肽粉20-30份、大豆多肽粉10-12份、桑叶提取物5-10份、栀子果油5-10份,可可粉5-10份、L-***糖5-10份、茯苓提取物3-5份、山楂提取物5-10份、营养酵母1-2份、胰酶2-5份、木糖醇5-8份。
优选的,所述营养酵母包括富硒酵母和/或富铬酵母。
优选的,所述胰酶包括胰蛋白酶、胰淀粉酶和胰脂肪酶,且按重量份计,胰蛋白酶:胰淀粉酶:胰脂肪酶=1:2:2。
优选的,所述苦瓜肽粉的制备方法包括如下步骤:
S11、取新鲜苦瓜、干燥苦瓜、苦瓜籽中的一项或几项作为苦瓜原料,加入苦瓜原料重量5倍的去离子水,并在25℃水温下浸泡10-12h,取出再用去离子水冲洗2-3遍;
S12、将冲洗后的苦瓜原料进行干燥,打碎磨浆,得到苦瓜浆液;
S13、取苦瓜浆液以及缓冲液进行混合,以获得混合体系,且按重量比计,苦瓜浆液:缓冲液=1:(3-5);记录混合体系总体积数值,将pH值调节为6.8-7后对混合体系进行温度处理,以获得浸提物;
所述温度处理过程包括:
升温至45-55℃,保温45-60min,再降温至20-25℃,保温25-30min,并记录此时混合体系整体的第一体积数值;按照(总体积-第一体积)*60%补入含有去离子水和缓冲液的第一混合液,且按照重量比计,去离子水:缓冲液=4:1;补入第一混合液后,升温至60-75℃,保温60-75min,再降温至45-55℃,保温30-35min,并记录此时混合体系整体的第二体积数值;按照(总体积-第二体积)*75%补入含有去离子水和缓冲液的第二混合液,且按照重量比计,去离子水:缓冲液=3:1;补入第二混合液后,升温至80-90℃,保温75-85min,再降温至60-75℃,保温35-45min;
S14、将浸提物温度降至20-25℃后对所述浸提物进行酶解,以获得苦瓜肽酶解体系;其中,所述酶解过程包括:
第一次酶解:将浸提物的pH值调节至7.5-8.5,按浸提物重量的5%加入胰蛋白酶,80-100转/min条件下搅拌,且搅拌的同时升温至35-40℃,保温45-60min后得到第一酶解体系;
第二次酶解:待第一酶解体系温度降至20-25℃后,再调整其pH值至3.0-4.0,按第一第一酶解体系重量的3%加入果胶酶,80-100转/min条件下搅拌,且搅拌的同时升温至45-55℃,保温40-60min后得到第二酶解体系;
第三次酶解:待第二酶解体系温度降至20-25℃后,再调整其pH值至4.5-5.0,按第二酶解体系重量的2%加入纤维素酶,80-100转/min条件下搅拌,且搅拌的同时升温至55-60℃,保温30-45min后得到第三酶解体系;
S15、待步骤S14中的酶解过程完成后,对获得的第三酶解体系升温至90℃,维持10min,以完成灭酶活过程,获得苦瓜肽粗提体系;再在所述苦瓜肽粗提体系中按其重量的4-5%加入活性炭,搅拌均匀,在65℃下保温60-90min后离心,去沉渣后获得苦瓜肽粗提液;
将所述苦瓜肽粗提液经硅藻土过滤,以得到苦瓜肽清液,且过滤压力为0.2-0.3MPa;再在所述苦瓜肽清液中按其重量加入4-5%的活性炭,静置45-50min后离心,去沉渣;
S16、将去沉渣后的苦瓜肽清液经过滤孔径为0.5-0.8μm的微滤陶瓷膜进行过滤,过滤温度为55-65℃,以获得微滤膜透过液;
再将所述微滤膜透过液经截留分子量为100-200kDa卷式超滤膜进行过滤,过滤温度为45-50℃,以获得超滤膜透过液;
再将所述超滤膜截留液经截留分子量为150-1000Da的卷式高压反渗透膜进行浓缩,以除去水分及部分残留无机盐和小分子杂质,浓缩温度为35-40℃,以得到苦瓜肽浓缩液;
S17、通过真空冷冻干燥方法对所述苦瓜肽浓缩液进行干燥,以获得苦瓜多肽蛋白含量不低于30%的苦瓜肽粉。
优选的,所述缓冲液为磷酸盐缓冲液。
优选的,所述栀子果油的提取方法包括:
S21、取新鲜栀子果放入水中,于25℃水温下浸泡24-36h后取出,用水冲洗2-3遍,烘干后磨碎,过100目筛,以获得栀子果粉;
S22、取栀子果粉,并加入其重量5-10倍的去离子水,以获得酶解原料,并对所述酶解原料进行酶解;其中,所述酶解过程包括:
第一次酶解:在所述酶解原料中按栀子果粉重量的5%加入胰蛋白酶以及按栀子果粉重量的45-55%加入用于调节细胞膜和/或细胞壁通透性的透性调节液,调节pH值至6.5-7.5,充分搅拌,且搅拌的同时升温至42-45℃,保温30-45min后得到第一酶解体系;所述透性调节液由酸溶液、甘油、氯化钠和溶菌酶组成,且按重量比计,酸溶液:甘油:氯化钠:溶菌酶=1:(0.7-1.0):(0.02-0.05):(0.03-0.06);
第二次酶解:待第一酶解体系温度降至20-25℃后,再调整其pH值至3.5-4.5,按第一体系重量的4%加入果胶酶,充分搅拌,且搅拌的同时升温至50-60℃,保温30-35min后得到第二酶解体系;
第三次酶解:待第二次酶解体系温度降至20-25℃后,再调整其pH值至4.0-5.5,按第二次酶解体系重量的3.5%加入纤维素酶,充分搅拌,且搅拌的同时升温至50-65℃,保温25-35min后得到第三酶解体系;
S23、待步骤S22中的酶解过程完成后,对获得的第三酶解体系升温至85℃,维持10min,以完成灭酶活过程,获得栀子果酶解体系;
S24、在所述栀子果酶解体系中按其重量的3%加入活性炭,搅拌均匀,在65℃下保温65-85min后离心,去沉渣后获得栀子果油粗提液;再将所述栀子果油粗提液经硅藻土过滤,得到栀子果油提取液,且过滤压力为0.3-0.4MPa;再在所述栀子果油提取液中按其重量加入3%的活性炭,静置45-50min后离心,去沉渣;再静置2-3h后取上层油层,即得到所述栀子果油。
还提供一种降低血糖血脂及糖化血红蛋白的多糖复合多肽的制备方法,其包括:
S100、制备苦瓜肽粉、栀子果油、桑叶提取物以及山楂提取物;所述桑叶提取物以及山楂提取物的制备方法相同,均包括:
(1)将原料放在其重量8-10倍的水中浸泡12-15h,然后加热至煮沸,煮沸状态持续1-2h后过滤,以获得第一滤液以及第一滤渣;
(2)对所述第一滤渣进行干燥,干燥后的第一过渣中加入其重量6-8倍的体积分数60%的乙醇,浸泡1-2h后加热至65℃-75℃,浸提1.5-2h,浸提过程中每10min搅拌一次,搅拌速率为200-300转/min;然后在8℃条件下静置24h,通过过滤分离获得第二滤液和第二滤渣;
(3)在第二滤渣中加入其重量8-10倍的体积分数60%的乙醇浸泡6-7h,加热至65℃-75℃,浸提2.5-3h,浸提过程中每10min搅拌一次,搅拌速率为200-300转/min,然后在8℃条件下静置24h,通过过滤分离获得第三滤液和第过滤渣;合并第一过滤液、第二过滤液以及第三过滤液,以获得提取物;
S200、称取如权利要求1所述重量份的燕麦膳食纤维粉、魔芋精粉、玉米须、苦瓜肽粉、大豆多肽粉、桑叶提取物、栀子果油、可可粉、茯苓提取物以及山楂提取物,充分混合,以获得原料混合物,再将所述原料混合物置于减压浓缩罐中,加入所述原料混合物重量5-8倍的去离子水,充分搅拌后加热至45-65℃进行真空减压浓缩,以获得浓缩液;
S300、将浓缩液置于配液罐中,再向配液罐内加入如权利要求1所述重量份的L-***糖、营养酵母、胰酶以及木糖醇,搅拌均匀后即获得所述具有降低血糖及糖化血红蛋白的多糖复合多肽。
本发明至少包括以下有益效果:
本发明通过阶段性升温、反复酶解以及多重过滤的提取工艺,使得苦瓜肽中苦瓜多肽蛋白含量不低于30%,进一步的,苦瓜肽与其他组分复配使用后,其具有明显的降血糖、降糖化血红蛋白等功效,并且可使得使用者摆脱使用化学药物降糖所带来的副作用。
本发明的其它优点、目标和特征将部分通过下面的说明体现,部分还将通过对本发明的研究和实践而为本领域的技术人员所理解。
具体实施方式
下面结合实例对本发明做进一步的详细说明,以令本领域技术人员参照说明书文字能够据以实施。
应当理解,本文所使用的诸如“具有”、“包含”以及“包括”术语并不配出一个或多个其它元件或其组合的存在或添加。
需要说明的是,下述实施方案中所述试验方法,如无特殊说明,均为常规方法,所述试剂和材料,如无特殊说明,均可从商业途径获得。
<实施例1>
按重量份计,本实施例中的降低血糖血脂及糖化血红蛋白的多糖复合多肽包括:燕麦膳食纤维粉20份、魔芋精粉11份、玉米须12份、苦瓜肽粉20份、大豆多肽10份、桑叶提取物6份、栀子果油6份,可可粉5份、L-***糖5份、茯苓提取物3份、山楂提取物6份、营养酵母1份、胰酶2份、木糖醇5份。其中,所述营养酵母包括富硒酵母和/或富铬酵母;所述胰酶包括胰酶含胰蛋白酶、胰淀粉酶和胰脂肪酶,且按重量份计,胰蛋白酶:胰淀粉酶:胰脂肪酶=1:2:2。
进一步的,所述苦瓜肽粉的制备方法包括如下步骤:
S11、取新鲜苦瓜、干燥苦瓜、苦瓜籽中的一项或几项作为苦瓜原料,加入苦瓜原料重量5倍的去离子水,并在25℃水温下浸泡10-12h(优选10.5h),取出再用去离子水冲洗2-3遍,以去除农残药残以及杂质;
S12、将冲洗后的苦瓜原料风干干燥,取出打碎磨浆,得到苦瓜浆液;
S13、取苦瓜浆液以及缓冲液(所述缓冲液为包含有酸、碱、盐等试剂的缓冲体系,如磷酸盐缓冲液)进行混合,以获得混合体系,按重量比计,苦瓜浆液:缓冲液=1:(3-5)(优选为1:4);记录混合体系总体积数值,将pH值调节为6.8-7后对混合体系进行温度处理,以获得浸提物;
其中,所述温度处理过程包括:
升温至45-55℃(优选为50℃),保温45-60min(优选为55min),降温至20-25℃(优选为22℃),保温25-30min(优选为28min),并记录此时混合体系整体的第一体积数值;因在前述升温、保温过程中,其反应体系内的水、酸等会被蒸发,有可能会导致其中酸、碱以及无机离子的溶解度产生变化,从而影响浸提效果,因此,在前述升温、保温过程后,需按照(总体积-第一体积)*60%补入含有去离子水和所述缓冲液的第一混合液,且按照重量比计,去离子水:缓冲液=4:1,由此对水、酸等蒸发后的反应体系进行补偿,使反应体系始终处于较优的浸提环境中;补入第一混合液后,升温至60-75℃(优选为65℃),保温60-75min(优选为65min),降温至45-55℃(优选为50℃),保温30-35min(优选为32℃),并记录此时混合体系整体的第二体积数值;按照(总体积-第二体积)*75%补入含有去离子水和缓冲液的第二混合液,且按照重量比计,去离子水:缓冲液=3:1,该次升温、保温的温度相较第一次而言有所提高,因此,反应体系内的水、酸等的蒸发效果更为明显,因此此次补入的第二混合液的比例要有所上升(增加到75%),且第二混合液中缓冲液的比例有所增加;补入第二混合液后,升温至80-90℃(优选为85℃),保温75-85min(优选为80min),降温至60-75℃(优选为70℃),保温35-45min(优选为40min);
本步骤中,通过阶段性的升温、保温,可使得组分的细胞结构(如细胞壁等)组成在不同的温度变化环境下被反复冲击、破坏,同时,每一升温、保温阶段结束后补充相应比例的水以及缓冲液,由此对水、酸等蒸发后的反应体系进行补偿,使反应体系始终处于较优的浸提环境中,以达到最佳的浸提效果;
S14、将浸提物温度降至20-25℃后对所述浸提物进行酶解,以获得苦瓜肽酶解体系;其中,所述酶解过程包括:
第一次酶解:将浸提物的pH值调节至7.5-8.5(优选为7.0),按浸提物重量的5%加入胰蛋白酶,80-100转/min条件下搅拌,且搅拌的同时升温至35-40℃(优选为37℃),保温45-60min(优选55min)后得到第一酶解体系;
第二次酶解:待第一酶解体系温度降至20-25℃后,再调整其pH值至3.0-4.0(优选为3.5),按第一第一酶解体系重量的3%加入果胶酶,80-100转/min条件下搅拌,且搅拌的同时升温至45-55℃(优选为50℃),保温40-60min(优选50min)后得到第二酶解体系;
第三次酶解:待第二酶解体系温度降至20-25℃后,再调整其pH值至4.5-5.0(优选为4.7),按第二酶解体系重量的2%加入纤维素酶,80-100转/min条件下搅拌,且搅拌的同时升温至55-60℃(优选为58℃),保温30-45min(优选35min)后得到第三酶解体系;
由于本发明中的组分多为植物组分,其细胞结构中包含有细胞壁,因此,本步骤中,通过在不同阶段采用不用的酶及酶解条件对细胞壁进行充分酶解,使细胞壁中的纤维素、果胶等成分破坏殆尽,可使得细胞壁内包含的有效成分(如苦瓜多肽蛋白)得以充分释放,以提高其提取效率;
S15、待步骤S14中的酶解过程完成后,对获得的第三酶解体系升温至90℃,维持10min,以完成灭酶活过程,获得苦瓜肽粗提体系;再在所述苦瓜肽粗提体系中按其重量的4-5%加入活性炭,搅拌均匀,在65℃下保温60-90min(优选75min)后离心,去沉渣后获得苦瓜肽粗提液;
将所述苦瓜肽粗提液经硅藻土过滤,以得到苦瓜肽清液,且过滤压力为0.2-0.3MPa(优选0.25MPa);再在所述苦瓜肽清液中按其重量加入4-5%的活性炭,静置45-50min后离心,去沉渣;
通过上述活性炭、硅藻土的吸附处理,苦瓜肽酶解液中的色素、悬浮颗粒以及胶体等杂质,使最后获得的成品纯度更高;
S16、将去沉渣后的苦瓜肽清液经过滤孔径为0.5-0.8μm的微滤陶瓷膜进行过滤,过滤温度为55-65℃(优选为60℃),以获得微滤膜透过液;进一步的,所述微滤陶瓷膜采用三支膜并联使用;
再将所述微滤膜透过液经截留分子量为100-200kDa卷式超滤膜进行过滤,除去大分子杂质,操作温度控制在55-65℃,以获得超滤膜透过液;其中,卷式超滤膜为能截留分子量为100-200kDa的卷式超滤膜,且所述卷式超滤膜采用两支膜并联使用;
再将所述超滤膜截留液经截留分子量为150-1000Da的卷式高压反渗透膜进行浓缩,以除去水分及部分残留无机盐和小分子杂质,浓缩温度在40℃以下,以得到苦瓜肽浓缩液,且苦瓜肽浓缩液的固形物含量≥40%;其中,卷式高压反渗透膜***为高压浓缩膜,具体由聚砜(PS)或聚醚砜(PFS)材料等复合材料膜制成,且采用四支膜串联使用;
该步骤中采用多层次的膜分离纯化技术分离纯化苦瓜多肽蛋白,其浓缩温度低,有效保证苦瓜蛋白多肽的天然活性及其高含量;
S17、通过真空冷冻干燥方法对所述苦瓜肽浓缩液进行干燥,以获得苦瓜多肽蛋白含量不低于30%的苦瓜肽粉。
此外,栀子果资源丰富、价廉易得、且含有多种功效,已在现代食品工业中发挥越来越重要的中作用、具体的,栀子果中主要含有黄酮类、环烯醚菇类、环烯米酮类等化合物,以及含有藏红花素、果胶、鞣质、多糖、藏红花酸、挥发油等成分,其中,黄酮类物质对高血压等疾病具有辅助治疗作用,可实现降血压、血糖等作用。因此,本实施例中还提供一种栀子果油的提取方法,其包括:
S21、取新鲜栀子果放入水中,于25℃水温下浸泡24-36h后取出,用水冲洗2-3遍,烘干后磨碎,过100目筛,以获得栀子果粉;
S22、取栀子果粉,并加入其重量5-10倍(优选8倍)的去离子水,以获得酶解原料,并对所述酶解原料进行酶解;其中,所述酶解过程包括:
第一次酶解:在所述酶解原料中按栀子果粉重量的5%加入胰蛋白酶以及按栀子果粉重量的45-55%(优选50%)加入用于调节细胞膜和/或细胞壁通透性的透性调节液,调节pH值至6.5-7.5(优选为7.0),充分搅拌,且搅拌的同时升温至42-45℃(优选为43.5℃),保温30-45min(优选为35min)后得到第一酶解体系;所述透性调节液由酸溶液、甘油、氯化钠和溶菌酶组成,且按重量比计,酸溶液:甘油:氯化钠:溶菌酶=1:(0.7-1.0):(0.02-0.05):(0.03-0.06)(优选为酸溶液:甘油:氯化钠:溶菌酶=1:0.8:0.03:0.04,且所述酸溶液为柠檬酸溶液);
第二次酶解:待第一酶解体系温度降至20-25℃后,再调整其pH值至3.5-4.5(优选为4.0),按第一体系重量的4%加入果胶酶,充分搅拌,且搅拌的同时升温至50-60℃(优选为55℃),保温30-35min(优选为32min)后得到第二酶解体系;
第三次酶解:待第二次酶解体系温度降至20-25℃后,再调整其pH值至4.0-5.5(优选为5.0),按第二次酶解体系重量的3.5%加入纤维素酶,充分搅拌,且搅拌的同时升温至50-65℃(优选为60℃),保温25-35min(优选为30min)后得到第三酶解体系;
S23、待步骤S22中的酶解过程完成后,对获得的第三酶解体系升温至85℃,维持10min,以完成灭酶活过程,获得栀子果酶解体系;
S24、在所述栀子果酶解体系中按其重量的3%加入活性炭,搅拌均匀,在65℃下保温65-85min(优选为75min)后离心,去沉渣后获得栀子果油粗提液;再将所述栀子果油粗提液经硅藻土过滤,得到栀子果油提取液,且过滤压力为0.3-0.4MPa(优选为0.35Mpa);再在所述栀子果油提取液中按其重量加入3%的活性炭,静置45-50min后离心,去沉渣;再静置2-3h(优选2.5h)后取上层油层,即得到所述栀子果油。
<实施例2>
本实施例与实施例1的不同之处仅在于,按重量份计,本实施例中的降低血糖血脂及糖化血红蛋白的多糖复合多肽由以下组分组成:燕麦膳食纤维粉25粉、魔芋精粉15份、玉米须14份、苦瓜肽粉28份、大豆多肽11份、桑叶提取物9份、栀子果油8份,可可粉9份、L-***糖10份、茯苓提取物5份、山楂提取物8份、营养酵母2份、胰酶4.5份、木糖醇7.5份、水200份。
<实施例3>
本实施例与实施例1的不同之处仅在于,按重量份计,本实施例中的降低血糖血脂及糖化血红蛋白的多糖复合多肽由以下组分组成:燕麦膳食纤维粉22粉、魔芋精粉13份、玉米须12.5份、苦瓜肽粉25份、大豆多肽11份、桑叶提取物8份、栀子果油8份,可可粉8份、L-***糖7份、茯苓提取物4份、山楂提取物8份、营养酵母1.5份、胰酶3.5份、木糖醇6.5份、水220份。
<苦瓜肽分子量检测结果>
采用申请号201710832199.8(“一种全程低温生产苦瓜多肽蛋白提取物的新方法、苦瓜多肽蛋白提取物及其应用”)的专利申请中实施例1所述的方法提取苦瓜肽,以作为对比例1,将其与通过本发明实施例1-3中的苦瓜肽粉制备方法制备获得的苦瓜肽粉进行高效凝胶过滤色谱法检测,以获得苦瓜肽分子量大小及分布范围,其结果如表1所示。
表1苦瓜肽分子量大小及分布范围
由此可见,本发明的苦瓜肽粉制备方法中,首先通过阶段性的升温、保温,可使得组分的细胞结构(如细胞壁等)组成在不同的温度变化环境下被反复冲击、破坏,同时,每一升温、保温阶段结束后补充相应比例的水以及缓冲液,由此对水、酸等蒸发后的反应体系进行补偿,使反应体系始终处于较优的浸提环境中,进一步再进行阶段性升温、反复酶解以及多层次的膜分离纯化技术提取工艺来制备苦瓜多肽蛋白,使得到的苦瓜多肽提取物中苦瓜多肽蛋白的含量高于30%,且提取物产品中不含大豆蛋白多肽等其他非苦瓜来源的多肽蛋白。从表1中可以看出,本发明制备获得的5000-7000Da范围内的苦瓜多肽片段占比接近30%,而5000-7000Da范围内的苦瓜多肽片段正是具有调节血糖功能、片段大小最接近胰岛素分子量的片段,因此得到的苦瓜多肽提取物具有非常好的调控血糖代谢效果,尤其是可大幅度提高胰岛素受体的结合能力及降血糖的效果。
<栀子果油检测结果>
采用申请号201110321487.X(“水酶法提取栀子油的方法”)的专利申请中实施例1所述的方法提取栀子果油,以作为对比例2,将其与通过本发明实施例1-3中的栀子果油提取方法获得的栀子果油进行检测,以获得藏花素、绿原酸、黄酮及栀子苷这几种主要降血糖、血脂有效成分的含量,其结果如表2所示。
表2栀子果油中藏花素、绿原酸、黄酮以及栀子苷含量
类似的,本发明中通过在不同阶段采用不用的酶及酶解条件对栀子果细胞壁进行充分酶解,使细胞壁中的纤维素、果胶等成分破坏殆尽,同时,酸溶液、甘油、氯化钠、溶菌酶可通过改变细胞壁或细胞膜结构来改变两者的通透性,因此本发明中通过采用调节细胞膜和/或细胞壁通透性的透性调节液来调节细胞膜和/或细胞壁透性,可使得细胞壁和/或细胞壁结构被破坏,使其内容的有效成分(如藏花素、黄酮类物质等)得以充分释放,进一步发挥其降血糖功效。
<实施例4>:
本实施例还提供了一种实施例1-3任一项所述降低血糖血脂及糖化血红蛋白的多糖复合多肽的制备方法,其包括:
S100、按照实施例1-3任一项中所述方法制备苦瓜肽粉和栀子果油,以及制备桑叶提取物以及山楂提取物;所述桑叶提取物以及山楂提取物的制备方法相同,均包括:
(1)将原料(即桑叶或山楂)放在其重量8-10倍的水中浸泡12-15h(优选13h),然后加热至煮沸,煮沸状态持续1-2h后过滤,以获得第一滤液以及第一滤渣;
(2)对所述第一滤渣进行干燥,干燥后的第一过渣中加入其重量6-8倍(优选7倍)的体积分数60%的乙醇,浸泡1-2h(优选1.5h)后加热至65℃-75℃(优选70℃),浸提1.5-2h,浸提过程中每10min搅拌一次,搅拌速率为200-300转/min;然后在8℃条件下静置24h,通过过滤分离获得第二滤液和第二滤渣;
(3)在第二滤渣中加入其重量8-10倍(优选9倍)的体积分数60%的乙醇浸泡6-7h(优选6.5h),加热至65℃-75℃(优选70℃),浸提2.5-3h,浸提过程中每10min搅拌一次,搅拌速率为200-300转/min,然后在8℃条件下静置24h,通过过滤分离获得第三滤液和第过滤渣;合并第一过滤液、第二过滤液以及第三过滤液,以获得提取物;
S200、称取如实施例1-3之一所述重量份的燕麦膳食纤维粉、魔芋精粉、玉米须、苦瓜肽粉、大豆多肽粉、桑叶提取物、栀子果油、可可粉、茯苓提取物以及山楂提取物,充分混合,以获得原料混合物,再将所述原料混合物置于减压浓缩罐中,加入所述原料混合物重量5-8倍的去离子水,充分搅拌后加热至45-65℃(优选55℃)进行真空减压浓缩,以获得浓缩液;
S300、将浓缩液置于配液罐中,再向配液罐内加入如实施例1-3之一所述重量份的L-***糖、营养酵母、胰酶以及木糖醇,搅拌均匀后即获得所述具有降低血糖及糖化血红蛋白的多糖复合多肽。
由此,通过对桑叶、山楂原料进行反复水提醇提,可使得其内部的活性成分(如桑叶多糖、桑叶生物碱、桑叶黄酮、山楂多糖等)充分释放,并且在第一次提取后对滤渣进行再次醇提,以最大限度提高对于原料的利用率,以此获得纯度更高的配比原料,使其活性成分能高效的发挥降血糖功效。
<降糖化血红蛋白功效评价试验>
选取体重为185~225g、健康的SD雄性大鼠,适应性喂养一周。将适应性喂养后的大鼠随机分为6组,每组20只。其中1组作为空白对照组,饲喂普通饲料,其余5组大鼠给予灌胃高脂乳剂10mL/kg,每天1次,连续1个月,末次灌胃后大鼠禁食不禁水12h,灌胃高脂乳剂的大鼠一次性腹腔注射链脲佐菌素STZ溶液25mg/kg建立糖尿病大鼠模型,注射链脲佐菌素72h后,大鼠禁食不禁水12h,尾部取血,检测空腹血糖,空腹血糖≥10.0mmol/L为造模成功的大鼠。空白对照组腹腔注射等剂量柠檬酸-柠檬酸钠缓冲液。
将造模成功的大鼠按血糖和体质量随机分为模型组、阳性对照组、低剂量组、中剂量组和高剂量组,每组20只大鼠。将实施例1-3中的具有降低血糖及糖化血红蛋白的多糖复合多肽(以下均为“多糖复合多肽”)用水稀释成灌胃药液,分别按照低剂量组给药量1.5g/kg、中剂量组给药量2.5g/kg和高剂量组给药量4.0g/kg灌胃,阳性对照组灌胃盐酸二甲双胍。每组每天灌胃1次,连续8周。检测前禁食不禁水12h,末次给药2h后,收集尿液,检测糖化血红蛋白含量和血脂含量,其结果如表3所示。以上各组动物除饲料配方有别外,自由饮水,不受限制。
表3多糖复合多肽对大鼠糖化血红蛋白含量和血脂含量影响
注:与空白对照组比较,**P<0.01;与模型组比较,#P<0.05,##P<0.01。
由表3可知,相比于空白对照组,模型组的血脂和糖化血红蛋白的含量均大幅升高(均增长也50%),说明糖尿病大鼠造模成功。与模型组相比,阳性对照组和给药组(高、中、低剂量组)的血脂和糖化血红蛋白的含量均有所下降,呈现显著性差异,其中,糖化血红蛋白和血脂含量以药物高剂量组下降更为明显(分别约下降40%和18%),表明本发明的多糖复合多肽对糖尿病有较好的治疗效果。
<降血糖功效评价试验>
选取健康的雄性小鼠,编号后在正常环境中适应性饲养2周,自由进食饮水。
适应性饲养结束后,随机分为6组,每组20只,其中一组为空白对照组,其余用剂量200mg/kg腹腔注射5%链脲佐菌素,2h之内如果有昏迷现象饲喂葡糖糖水直至清醒,2天以后,测定空腹血糖,取血糖值在10以上的作为糖尿病动物模型。
将造模成功的小鼠按血糖和体质量随机分为模型组、阳性对照组、低剂量组、中剂量组和高剂量组,每组20只小鼠。将实施例1-3中的具有降低血糖及糖化血红蛋白的多糖复合多肽用水稀释成注射药液,分别按照低剂量组给药量1.0g/kg、中剂量组给药量2.0g/kg和高剂量组给药量3.0g/kg进行腹腔注射给药,阳性对照组腹腔注射盐酸二甲双胍。每组每天腹腔注射1次,连续15天。15天后对小鼠禁食8h测定空腹血糖值和尿糖值,其结果如表4所示。以上各组动物除饲料配方有别外,自由饮水,不受限制。
实验过程中每天对各个组份的小鼠进行腹腔注射给药,连续15天,45天后,对小鼠禁食8h测定空腹血糖值。实验结束,记录最终血糖含量。
表4多糖复合多肽对小鼠尿糖和空腹血糖值影响
由表4可知,与模型组相比,阳性对照组和给药组(高、中、低剂量组)的小鼠尿糖和空腹血糖值均有所下降,呈现显著性差异,其中,相对于模型组,高、中、低剂量组的尿糖分别下降9%、32%和56%,空腹血糖分别下降21%、39%和52%,高剂量组的能显著降低尿糖和空腹血糖含量。
<降血脂功效评价试验>
选取健康的雄性大鼠,编号后在正常环境中适应性饲养2周,自由进食饮水。适应性饲养结束后,随机分为6组,每组10只,分别为空白对照组、高脂模型组、阳性治疗组、高剂量组、中剂量组、低剂量组。除空白对照组外,其余各组均采用高脂饲料。将本发明的多糖复合多肽以及辛伐他汀配制成相应浓度的溶液,阳性对照组按照50mg/kgbw·d灌胃辛伐他汀溶液,高、中、低剂量组分别按照100mg/kg bw·d、50mg/kg bw·d和25mg/kg bw·d灌胃多糖复合多肽溶液。其他组灌胃相应体积蒸馏水。治疗期为4周,连续喂养10周后,末次喂养后。禁食不禁水10h,主动脉取血测总胆固醇(TC)和甘油三酯(TG)指标其结果如表5所示。
表5多糖复合多肽对大鼠TC和TG含量影响
注:与空白组对照比较,*p<0.05;与高脂组相比,#p<0.05,##p<0.01。
由表5可知,与高脂组相比,给药组(高、中、低剂量组)的大鼠总胆固醇(TC)和甘油三酯(TG)含量均显著下降,因此本发明中的多糖复合多肽具有良好的降血脂效果。
需要说明的是,上述实施例1至4中的技术方案可进行任意组合,且组合后获得的技术方案均属于本发明的保护范围。
综上所述,本发明通过阶段性升温、反复酶解以及多重过滤的提取工艺,使得苦瓜肽中苦瓜多肽蛋白含量不低于30%,进一步的,苦瓜肽与其他组分复配使用后,其具有明显的降血糖、降糖化血红蛋白等功效,并且可使得使用者摆脱使用化学药物降糖所带来的副作用。
这里说明的设备数量和处理规模是用来简化本发明的说明的。对本发明的应用、修改和变化对本领域的技术人员来说是显而易见的。
尽管本发明的实施方案已公开如上,但其并不仅仅限于说明书和实施方式中所列运用,它完全可以被适用于各种适合本发明的领域,对于熟悉本领域的人员而言,可容易地实现另外的修改,因此在不背离权利要求及等同范围所限定的一般概念下,本发明并不限于特定的细节和这里示出与描述的实例。
Claims (7)
1.降低血糖血脂及糖化血红蛋白的多糖复合多肽,其特征在于,按重量份计,其包括:燕麦膳食纤维粉20-25份、魔芋精粉10-15份、玉米须10-15份、苦瓜肽粉20-30份、大豆多肽粉10-12份、桑叶提取物5-10份、栀子果油5-10份,可可粉5-10份、L-***糖5-10份、茯苓提取物3-5份、山楂提取物5-10份、营养酵母1-2份、胰酶2-5份、木糖醇5-8份。
2.如权利要求1所述的多糖复合多肽,其特征在于,所述营养酵母包括富硒酵母和/或富铬酵母。
3.如权利要求1所述的多糖复合多肽,其特征在于,所述胰酶包括胰蛋白酶、胰淀粉酶和胰脂肪酶,且按重量份计,胰蛋白酶:胰淀粉酶:胰脂肪酶=1:2:2。
4.如权利要求1所述的多糖复合多肽,其特征在于,所述苦瓜肽粉的制备方法包括如下步骤:
S11、取新鲜苦瓜、干燥苦瓜、苦瓜籽中的一项或几项作为苦瓜原料,加入苦瓜原料重量5倍的去离子水,并在25℃水温下浸泡10-12h,取出再用去离子水冲洗2-3遍;
S12、将冲洗后的苦瓜原料进行干燥,打碎磨浆,得到苦瓜浆液;
S13、取苦瓜浆液以及缓冲液进行混合,以获得混合体系,且按重量比计,苦瓜浆液:缓冲液=1:(3-5);记录混合体系总体积数值,将pH值调节为6.8-7后对混合体系进行温度处理,以获得浸提物;
所述温度处理过程包括:
升温至45-55℃,保温45-60min,再降温至20-25℃,保温25-30min,并记录此时混合体系整体的第一体积数值;按照(总体积-第一体积)*60%补入含有去离子水和缓冲液的第一混合液,且按照重量比计,去离子水:缓冲液=4:1;补入第一混合液后,升温至60-75℃,保温60-75min,再降温至45-55℃,保温30-35min,并记录此时混合体系整体的第二体积数值;按照(总体积-第二体积)*75%补入含有去离子水和缓冲液的第二混合液,且按照重量比计,去离子水:缓冲液=3:1;补入第二混合液后,升温至80-90℃,保温75-85min,再降温至60-75℃,保温35-45min;
S14、将浸提物温度降至20-25℃后对所述浸提物进行酶解,以获得苦瓜肽酶解体系;其中,所述酶解过程包括:
第一次酶解:将浸提物的pH值调节至7.5-8.5,按浸提物重量的5%加入胰蛋白酶,80-100转/min条件下搅拌,且搅拌的同时升温至35-40℃,保温45-60min后得到第一酶解体系;
第二次酶解:待第一酶解体系温度降至20-25℃后,再调整其pH值至3.0-4.0,按第一第一酶解体系重量的3%加入果胶酶,80-100转/min条件下搅拌,且搅拌的同时升温至45-55℃,保温40-60min后得到第二酶解体系;
第三次酶解:待第二酶解体系温度降至20-25℃后,再调整其pH值至4.5-5.0,按第二酶解体系重量的2%加入纤维素酶,80-100转/min条件下搅拌,且搅拌的同时升温至55-60℃,保温30-45min后得到第三酶解体系;
S15、待步骤S14中的酶解过程完成后,对获得的第三酶解体系升温至90℃,维持10min,以完成灭酶活过程,获得苦瓜肽粗提体系;再在所述苦瓜肽粗提体系中按其重量的4-5%加入活性炭,搅拌均匀,在65℃下保温60-90min后离心,去沉渣后获得苦瓜肽粗提液;
将所述苦瓜肽粗提液经硅藻土过滤,以得到苦瓜肽清液,且过滤压力为0.2-0.3MPa;再在所述苦瓜肽清液中按其重量加入4-5%的活性炭,静置45-50min后离心,去沉渣;
S16、将去沉渣后的苦瓜肽清液经过滤孔径为0.5-0.8μm的微滤陶瓷膜进行过滤,过滤温度为55-65℃,以获得微滤膜透过液;
再将所述微滤膜透过液经截留分子量为100-200kDa卷式超滤膜进行过滤,过滤温度为45-50℃,以获得超滤膜透过液;
再将所述超滤膜截留液经截留分子量为150-1000Da的卷式高压反渗透膜进行浓缩,以除去水分及部分残留无机盐和小分子杂质,浓缩温度为35-40℃,以得到苦瓜肽浓缩液;
S17、通过真空冷冻干燥方法对所述苦瓜肽浓缩液进行干燥,以获得苦瓜多肽蛋白含量不低于30%的苦瓜肽粉。
5.如权利要求4所述的多糖复合多肽,其特征在于,所述缓冲液为磷酸盐缓冲液。
6.如权利要求1所述的多糖复合多肽,其特征在于,所述栀子果油的提取方法包括:
S21、取新鲜栀子果放入水中,于25℃水温下浸泡24-36h后取出,用水冲洗2-3遍,烘干后磨碎,过100目筛,以获得栀子果粉;
S22、取栀子果粉,并加入其重量5-10倍的去离子水,以获得酶解原料,并对所述酶解原料进行酶解;其中,所述酶解过程包括:
第一次酶解:在所述酶解原料中按栀子果粉重量的5%加入胰蛋白酶以及按栀子果粉重量的45-55%加入用于调节细胞膜和/或细胞壁通透性的透性调节液,调节pH值至6.5-7.5,充分搅拌,且搅拌的同时升温至42-45℃,保温30-45min后得到第一酶解体系;所述透性调节液由酸溶液、甘油、氯化钠和溶菌酶组成,且按重量比计,酸溶液:甘油:氯化钠:溶菌酶=1:(0.7-1.0):(0.02-0.05):(0.03-0.06);
第二次酶解:待第一酶解体系温度降至20-25℃后,再调整其pH值至3.5-4.5,按第一体系重量的4%加入果胶酶,充分搅拌,且搅拌的同时升温至50-60℃,保温30-35min后得到第二酶解体系;
第三次酶解:待第二次酶解体系温度降至20-25℃后,再调整其pH值至4.0-5.5,按第二次酶解体系重量的3.5%加入纤维素酶,充分搅拌,且搅拌的同时升温至50-65℃,保温25-35min后得到第三酶解体系;
S23、待步骤S22中的酶解过程完成后,对获得的第三酶解体系升温至85℃,维持10min,以完成灭酶活过程,获得栀子果酶解体系;
S24、在所述栀子果酶解体系中按其重量的3%加入活性炭,搅拌均匀,在65℃下保温65-85min后离心,去沉渣后获得栀子果油粗提液;再将所述栀子果油粗提液经硅藻土过滤,得到栀子果油提取液,且过滤压力为0.3-0.4MPa;再在所述栀子果油提取液中按其重量加入3%的活性炭,静置45-50min后离心,去沉渣;再静置2-3h后取上层油层,即得到所述栀子果油。
7.一种降低血糖血脂及糖化血红蛋白的多糖复合多肽的制备方法,其特征在于,包括:
S100、制备苦瓜肽粉、栀子果油、桑叶提取物以及山楂提取物;所述桑叶提取物以及山楂提取物的制备方法相同,均包括:
(1)将原料放在其重量8-10倍的水中浸泡12-15h,然后加热至煮沸,煮沸状态持续1-2h后过滤,以获得第一滤液以及第一滤渣;
(2)对所述第一滤渣进行干燥,干燥后的第一过渣中加入其重量6-8倍的体积分数60%的乙醇,浸泡1-2h后加热至65℃-75℃,浸提1.5-2h,浸提过程中每10min搅拌一次,搅拌速率为200-300转/min;然后在8℃条件下静置24h,通过过滤分离获得第二滤液和第二滤渣;
(3)在第二滤渣中加入其重量8-10倍的体积分数60%的乙醇浸泡6-7h,加热至65℃-75℃,浸提2.5-3h,浸提过程中每10min搅拌一次,搅拌速率为200-300转/min,然后在8℃条件下静置24h,通过过滤分离获得第三滤液和第过滤渣;合并第一过滤液、第二过滤液以及第三过滤液,以获得提取物;
S200、称取如权利要求1所述重量份的燕麦膳食纤维粉、魔芋精粉、玉米须、苦瓜肽粉、大豆多肽粉、桑叶提取物、栀子果油、可可粉、茯苓提取物以及山楂提取物,充分混合,以获得原料混合物,再将所述原料混合物置于减压浓缩罐中,加入所述原料混合物重量5-8倍的去离子水,充分搅拌后加热至45-65℃进行真空减压浓缩,以获得浓缩液;
S300、将浓缩液置于配液罐中,再向配液罐内加入如权利要求1所述重量份的L-***糖、营养酵母、胰酶以及木糖醇,搅拌均匀后即获得所述具有降低血糖及糖化血红蛋白的多糖复合多肽。
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