CN114028534B - 一种治疗糖尿病的低分子量苦瓜多肽含片的制备方法 - Google Patents
一种治疗糖尿病的低分子量苦瓜多肽含片的制备方法 Download PDFInfo
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Abstract
本发明涉及治疗糖尿病的低分子量苦瓜多肽含片的制备方法,可有效解决既提高苦瓜多肽得率和复溶率,又保证舌下吸收快,顺应性好,提高疗效的问题,方法是,苦瓜籽预处理,采用米曲霉进行发酵处理苦瓜籽,发酵完成后,将发酵料干燥,粉碎成粉末,用乙醇提取,离心后收集过滤,蒸馏回收乙醇;加水后用氨水调pH值至9.6‑9.8,离心,取上清液;用酸调pH值至3.5,加入无水乙醇,进行沉淀,收集沉淀,再用无水乙醇洗脱后,进行干燥,粉碎,按常规制片方法加入辅料,压制成片,得低分子量苦瓜多肽含片。本发明方法科学合理,易操作,苦瓜多肽得率高,纯度高,所制成的含片吸收快,顺应性好,具有显著的降糖功能,可有效用于治疗糖尿病。
Description
技术领域
本发明涉及医药,特别是一种治疗糖尿病的低分子量苦瓜多肽含片的制备方法。
背景技术
糖尿病是一组以高血糖为特征的代谢性疾病。高血糖则是由于胰岛素分泌缺陷或其生物作用受损,或两者兼有引起。糖尿病时长期存在的高血糖,导致各种组织,特别是眼、肾、心脏、血管、神经的慢性损害、功能障碍。近几十年中,全球糖尿病患者的人数以惊人的速度迅速增长。据统计,2007年全球约2.46亿人患有糖尿病,预计到2030年,糖尿病患者将增长到3.8亿,且大部分集中于发展中国家。预计中国受糖尿病影响的人群达到9300万,目前已成为影响中国人身心健康的主要公共卫生问题。2002年中国无并发症Ⅱ型糖尿病的年人均直接医疗成本为13833.3元,全国城市Ⅱ型糖尿病及其并发症的年总成本为233.8亿元,其中,直接医疗成本为187.5亿元,占全国医疗卫生总费用的3.94%,直接非医疗成本为22.8亿元,间接成本为23.5亿元。
糖尿病给社会和家庭都带来了沉重的经济负担。针对糖尿病的发病机理情况,国外开发的许多西药存在一定的副作用。目前,口服药物主要包括磺脲类、双胍类、α-葡萄糖苷酶抑制剂、胰岛素分泌促进剂、胰岛素增敏剂等,使用胰岛素,不易控制给药量,且长期使用会产生胰岛素抵抗。α-葡萄糖苷酶抑制剂最常见的不良反应是肠胃道副作用,如腹部不适、胀气、排气、腹泻等。磺脲类药物常见的不良反应有:食欲减退、恶心、呕吐、腹泻及腹痛等肠胃反应;皮肤瘙痒、红斑、寻麻疹、麻疹样皮疹或斑丘疹等皮肤反应;白细胞减少、粒细胞缺乏、血小板减少、溶血性贫血、再生不良性贫血及全血细胞减少等血液系统疾病;及中毒性肝炎等。使用胰岛素增敏剂药物,如曲格列酮,使心率危险性增加,并且对肝脏具有不同程度的毒性。
因此,如何进行糖尿病的防治保健,寻找安全、有效的治疗糖尿病的新功能物质,特别是从天然资源中去筛选和研究降糖成分,进行绿色降糖,已成为国内外医药工作者们所关注的重要课题和迫在眉睫的任务。在各种药食同源植物资源中,苦瓜作为一种极具开发潜力和开发价值的植物,引起了食品界和医学界专家们极大的兴趣。现虽有一种苦瓜多肽含片治疗糖尿病,但由于制备方法上存在的问题,含片中多肽分子量大,舌下吸收慢,需长时间含服,服药顺应性差,其疗效不尽人意,而且制备的苦瓜多肽得率低,复溶率低,不利于充分利用资源,因此,苦瓜多肽含片制备方法上的改进和创新势在必行。
发明内容
针对上述情况,为克服现有技术之缺陷,本发明之目的就是提供一种治疗糖尿病的低分子量苦瓜多肽含片的制备方法,可有效解决既提高苦瓜多肽得率和复溶率,又保证舌下吸收快,顺应性好,提高疗效的问题。
本发明解决的技术方案是,一种治疗糖尿病的低分子量苦瓜多肽含片的制备方法,包括以下步骤:
a)苦瓜籽预处理,采用米曲霉进行发酵处理苦瓜籽,发酵完成后,将发酵料干燥,粉碎成粉末;
b)采用质量含量为83%-87%的乙醇溶液提取,离心后收集过滤,蒸馏回收乙醇;
c)加水后用氨水调pH值至9.6-9.8,离心,取上清液;
d)用酸调pH值至3.5,加入无水乙醇,进行沉淀,收集沉淀,再用无水乙醇洗脱后,进行干燥,粉碎,按常规制片方法加入辅料,压制成片,得低分子量苦瓜多肽含片。
本发明方法科学合理,易操作,苦瓜多肽得率高,纯度高,所制成的含片吸收快,顺应性好,具有显著的降糖功能,可有效用于治疗糖尿病,是治疗糖尿病药物上的一大创新。
具体实施方式
以下结合具体情况和实施对本发明的具体实施方式作详细说明。
实施例1
本发明一种治疗糖尿病的低分子量苦瓜多肽含片的制备方法,包括以下步骤:
1)苦瓜籽预处理
选取新鲜的苦瓜籽或无菌水清洗后浸泡6h的干燥苦瓜籽50kg,碾碎成颗粒,颗粒大小为0.5-1.5mm,进行预处理;
2)发酵苦瓜籽的制备
a)菌种活化:将米曲霉菌种接种于经过无菌验证的土豆培养基的试管斜面或平板中,土豆培养基是,马铃薯200g、葡萄糖20g、琼脂 15~20g,加蒸馏水1000mL制成(公知技术),然后将接种后的斜面或平板在26~30℃下培养72h,得活化米曲霉菌种;
b)菌种扩培:取500mL的锥形瓶,添加液体培养基200mL,每升液体培养基中含有苦瓜籽粉10-30g、蛋白胨50g、苦瓜籽油2-8mL、硝酸钠2g、磷酸氢二钾1g、氯化钠0.5g和硫酸亚铁0.01g,将液体培养基进行湿热灭菌,灭菌温度121℃,时间为15min;培养基温度降到23-27℃,移至超净工作台,接入活化米曲霉菌种,在26-30℃恒温摇床中培养,摇床振荡频率为200r/min,培养时间为36h,得扩培菌种;
c)苦瓜籽发酵:苦瓜籽料与扩培菌种的质量体积比为18-22︰1 g/mL,质量体积是指固体质量以g计,液体以mL计,进行发酵,温度28-32℃,湿度85-95%,发酵48-72h,发酵期间每8h翻拌一次,再将发酵料干燥,粉碎,得干燥的发酵苦瓜籽粉;
3)乙醇提取
加质量浓度83-87%的乙醇溶液250L,于20-24℃下浸泡提取30-36h,离心、过滤,滤液蒸馏回收乙醇,加100L水,用氨水调pH值至9.6-9.8,离心,得上清液;
4)多肽沉淀
将上清液用1N的盐酸调pH值至3.5,加入90-110L无水乙醇,于4℃下放置16小时,得沉淀;
5)干燥粉碎
沉淀加无水乙醇溶解脱洗2-3次,每次10-20min,回收乙醇,收集固体沉淀,于42-45℃进行真空干燥,粉碎,过 100 目筛,得苦瓜多肽;
6)制片:首先,将木糖醇过100目筛,过18目筛网制粒,湿颗粒于 40℃以下干燥,干颗粒过 16 目筛整粒,称重,再按常规制片方法加入苦瓜多肽和其余辅料,混匀,确定片重,压制成片,即成治疗糖尿病的低分子量苦瓜多肽含片;
所述的其余辅料为微粉硅胶、香精和柠檬酸。
实施例2
本发明一种治疗糖尿病的低分子量苦瓜多肽含片的制备方法,包括以下步骤:
1)苦瓜籽预处理
选取新鲜的苦瓜籽50kg,碾碎成颗粒,颗粒大小为1.0mm,进行预处理;
2)发酵苦瓜籽的制备
a)菌种活化:将米曲霉菌种接种于经过无菌验证的土豆培养基的试管斜面或平板中,然后将接种后的斜面或平板在28℃下培养72h,得活化米曲霉菌种;
b)菌种扩培:取500mL的锥形瓶,添加液体培养基200mL,每升液体培养基中含有苦瓜籽粉20g、蛋白胨50g、苦瓜籽油5mL、硝酸钠2g、磷酸氢二钾1g、氯化钠0.5g和硫酸亚铁0.01g,将液体培养基进行湿热灭菌,灭菌温度121℃,时间为15min;培养基温度降到25℃,移至超净工作台,接入活化米曲霉菌种,在28℃恒温摇床中培养,摇床振荡频率为200r/min,培养时间为36h,得扩培菌种;
c)苦瓜籽发酵:苦瓜籽料与扩培菌种的质量体积比为20︰1 g/mL,进行发酵,温度28℃,湿度85%,发酵60h,发酵期间每8h翻拌一次,再将发酵料干燥,粉碎,得干燥的发酵苦瓜籽粉;
3)乙醇提取
加质量浓度85%的乙醇溶液250L,于22℃下浸泡提取34h,离心、过滤,滤液蒸馏回收乙醇,加100L水,用氨水调pH值至9.7,离心,得上清液;
4)多肽沉淀
将上清液用1N的盐酸调pH值至3.5,加入100L无水乙醇,于4℃下放置16小时,得沉淀;
5)干燥粉碎
沉淀加无水乙醇溶解脱洗2次,每次20min,回收乙醇,收集固体沉淀,于44℃进行真空干燥,粉碎,过 100 目筛,得苦瓜多肽;
6)制片:首先,将木糖醇过100目筛,过18目筛网制粒,湿颗粒于 40℃以下干燥,干颗粒过 16 目筛整粒,称重,再按常规制片方法加入苦瓜多肽和其余辅料,混匀,确定片重,压制成片,即成治疗糖尿病的低分子量苦瓜多肽含片。
实施例3
本发明一种治疗糖尿病的低分子量苦瓜多肽含片的制备方法,包括以下步骤:
1)苦瓜籽预处理
选取新鲜的苦瓜籽50kg,碾碎成颗粒,颗粒大小为1.5mm,进行预处理;
2)发酵苦瓜籽的制备
a)菌种活化:将米曲霉菌种接种于经过无菌验证的土豆培养基的试管斜面或平板中,然后将接种后的斜面或平板在28℃下培养72h,得活化米曲霉菌种;
b)菌种扩培:取500mL的锥形瓶,添加液体培养基200mL,每升液体培养基中含有苦瓜籽粉20g、蛋白胨50g、苦瓜籽油5mL、硝酸钠2g、磷酸氢二钾1g、氯化钠0.5g和硫酸亚铁0.01g,将液体培养基进行湿热灭菌,灭菌温度121℃,时间为15min;培养基温度降到23-27℃,移至超净工作台,接入活化米曲霉菌种,在27℃恒温摇床中培养,摇床振荡频率为200r/min,培养时间为36h,得扩培菌种;
c)苦瓜籽发酵:苦瓜籽料与扩培菌种的质量体积比为20︰1 g/mL,质量体积是指固体质量以g计,液体以mL计,进行发酵,温度28℃,湿度85%,发酵72h,发酵期间每8h翻拌一次,再将发酵料干燥,粉碎,得干燥的发酵苦瓜籽粉;
3)乙醇提取
加质量浓度87%的乙醇溶液250L,于24℃下浸泡提取30h,离心、过滤,滤液蒸馏回收乙醇,加100L水,用氨水调pH值至9.8,离心,得上清液;
4)多肽沉淀
将上清液用1N的盐酸调pH值至3.5,加入90L无水乙醇,于4℃下放置16小时,得沉淀;
5)干燥粉碎
沉淀加无水乙醇溶解脱洗3次,每次10min,回收乙醇,收集固体沉淀,于45℃进行真空干燥,粉碎,过 100 目筛,得苦瓜多肽;
6)制片:首先,将木糖醇过100目筛,过18目筛网制粒,湿颗粒于 40℃以下干燥,干颗粒过 16 目筛整粒,称重,再按常规制片方法加入苦瓜多肽和其余辅料,混匀,确定片重,压制成片,即成治疗糖尿病的低分子量苦瓜多肽含片。
实施例4
本发明一种治疗糖尿病的低分子量苦瓜多肽含片的制备方法,包括以下步骤:
1)苦瓜籽预处理
选取无菌水清洗后浸泡6h的干燥苦瓜籽50kg,碾碎成颗粒,颗粒大小为0.5mm,进行预处理;
2)发酵苦瓜籽的制备
a)菌种活化:将米曲霉菌种接种于经过无菌验证的土豆培养基的试管斜面或平板中,然后将接种后的斜面或平板在28℃下培养72h,得活化米曲霉菌种;
b)菌种扩培:取500mL的锥形瓶,添加液体培养基200mL,每升液体培养基中含有苦瓜籽粉20g、蛋白胨50g、苦瓜籽油5mL、硝酸钠2g、磷酸氢二钾1g、氯化钠0.5g和硫酸亚铁0.01g,将液体培养基进行湿热灭菌,灭菌温度121℃,时间为15min;培养基温度降到25℃,移至超净工作台,接入活化米曲霉菌种,在30℃恒温摇床中培养,摇床振荡频率为200r/min,培养时间为36h,得扩培菌种;
c)苦瓜籽发酵:苦瓜籽料与扩培菌种的质量体积比为20︰1 g/mL,进行发酵,温度28℃,湿度90%,发酵48h,发酵期间每8h翻拌一次,再将发酵料干燥,粉碎,得干燥的发酵苦瓜籽粉;
3)乙醇提取
加质量浓度83%的乙醇溶液250L,于20℃下浸泡提取36h,离心、过滤,滤液蒸馏回收乙醇,加100L水,用氨水调pH值至9.6,离心,得上清液;
4)多肽沉淀
将上清液用1N的盐酸调pH值至3.5,加入110L无水乙醇,于4℃下放置16小时,得沉淀;
5)干燥粉碎
沉淀加无水乙醇溶解脱洗2次,每次15min,回收乙醇,收集固体沉淀,于42℃进行真空干燥,粉碎,过 100 目筛,得苦瓜多肽;
6)制片:首先,将木糖醇过100目筛,过18目筛网制粒,湿颗粒于 40℃以下干燥,干颗粒过 16 目筛整粒,称重,再按常规制片方法加入苦瓜多肽和其余辅料,混匀,确定片重,压制成片,即成治疗糖尿病的低分子量苦瓜多肽含片。
本发明方法与现有方法制备的产品相比,多肽得率高,纯度高,分子量低,复溶率高,所制成的含片产品口感好,吸收快,顺应性好,降糖效果好,可有效用于治疗糖尿病,并经实验取得了非常好的有益技术效果,有关资料如下:
一、多肽含量的测定:
采用双缩脲法,以牛血清白蛋白V为标准绘制标准曲线,精确称取牛血清白蛋白V0.5000g,配制成5mg/mL标准蛋白溶液,取6支试管,编号1-6,一次加入标准蛋白溶液0、0.4、0.8、1.2、1.6、2.0mL,加蒸馏水补足至2.0mL,即蛋白质含量分别为0、2.0、4.0、6.0、8.0、10.0,各加入4.0mL双缩脲试剂,混匀后室温静置15min,于540nm下测定其吸光值,根据吸光值(y)与蛋白质含量(x)绘制标准曲线。建立的回归方程为y=4.028+0.0428,R2=0.9987。根据得到的多肽含量计算苦瓜多肽纯度和得率。采用SDS-PAGE凝胶电泳对比marker测定苦瓜多肽主条带分子量分布范围并记录。试验结果如表1所示。
表1不同实施例多肽含量的得率
组别 | 得率(%) | 纯度(%) | 苦瓜多肽主条带分子量分布范围kDa | 复溶率(%) |
实施例2组 | 15.23 | 98.2 | 5-6 | 98.6 |
实施例3组 | 14.86 | 98.2 | 5-6 | 98.6 |
实施例4组 | 15.02 | 98.1 | 5-6 | 98.5 |
对比例组 | 6.52 | 95.4 | 9-11 | 52.4 |
对比例组为现有方法,步骤是:
1)苦瓜籽预处理
选取新鲜干净的苦瓜籽50kg,将新鲜苦瓜籽碾碎并粉碎成粉末。
2)乙醇提取
加250LpH值为9.5的75%的乙醇溶液,于22℃下浸泡提取24h,离心后过滤。滤液蒸馏回收乙醇,加100L水,用氨水调pH值至9.5,离心;
3)丙酮提取
取上清液,用1N的盐酸调pH值至3.5,加入45L丙酮,于4℃下放置16h,沉淀;
5)干燥粉碎
加适量丙酮脱洗;收集固体沉淀,于42℃以下进行真空干燥,干燥后粉碎,过100目筛,制得苦瓜多肽。
6)压片:木糖醇过100目筛,以常规制药方法,用18目筛网制粒,湿颗粒于 40℃以下干燥,干颗粒过16目筛,进行整粒。称重,按处方比例加入苦瓜提取物、微粉硅胶、香精、柠檬酸,混匀,确定片重,压片。
二、复溶率测定:
称取实施例2-4和对比例制备的苦瓜多肽,放入试管中(每个样品三个平行实验),加入用氨水调至pH为9.0的水5mL,超声震荡3min,4000rpm离心10min,倒去上清液,烘干试管至恒重,减去试管质量得到为溶解的苦瓜多肽质量,计算复溶率。试验结果如表1所示。
表1测定结果表明,得实施例2-4制备的苦瓜多肽主条带分子量分布在5-6kDa,经检测其具有较高的降血糖活性。从复溶率的结果可以看出,丙酮对苦瓜多肽复溶率影响较大。
三、动物喂养实验
选用离乳大鼠40只,雌雄各半,按体重随机分为四组,即本发明低剂量(10mg/kg)、本发明中剂量(100mg/kg)、本发明高剂量(300mg/kg)组和阴性对照组。连续30天按剂量设计灌胃不同剂量的实施例2制备的苦瓜多肽给予大鼠,阴性对照组给予蒸馏水,灌胃量1mL/kg。试验期间,动物自由摄食和饮水,每天观察并记录动物的一般表现、行为、中毒症状和死亡情况。第15、30天程一次体重并记录。第30天禁食16h,第31天取血测定血红蛋白、红细胞计数、白细胞分类等血液学指标,实验结果见表2:
表2苦瓜多肽对正常大鼠体重的影响
表2结果可见,各组实验动物的初始、中期和结束体重均无显著性差异,说明实施例1制备的苦瓜多肽对正常大鼠体重无影响。
表3苦瓜多肽组分对正常大鼠空腹血糖的影响
*:与正常对照组相比,差异显著(p<0.05)
表3表明各组动物控股血糖值均无明显差异,说明实施例2苦瓜多肽对正常大鼠无影响。
四、降糖活性试验
各组分对糖尿病小鼠血糖的影响:大鼠50只,取10只小鼠作为正常对照组其余的大鼠以200mg/kg体重剂量腹腔注射4%的四氧嘧啶水溶液,建立糖尿病模型。将模型大鼠分为4组,每组10只,其中一组为模型对照组,口腔灌胃蒸馏水。另4组(实施例2组、实施例3组、对比例组)为实验组,以25mg/Kg体重的多肽剂量分别口腔灌胃多肽浓度为1.5mg/mL的各超滤组分溶液,连续3天,每天1次。第3天口腔灌胃3h后,尾部取血,测血糖值,结果见表4:
表4苦瓜超滤组分对四氧嘧啶糖尿病小鼠血糖的影晌
组别 | 实验动物/只 | 血糖值(mmol/L) |
正常对照组 | 10 | 6.04±0.36 |
模型对照组 | 10 | 24.58±1.60 |
实施例2组 | 10 | 20.52±0.68* |
实施例3组 | 10 | 21.36±0.82* |
对比例组 | 10 | 23.48±0.78 |
*:与模型对照组相比,差异显著(p<0.05)
表4表明实施例2、3制备的苦瓜多肽具有显著降糖尿病模型小鼠的血糖(p<0.05)功能。对比例有降糖趋势。
在对上述实施例进行实验时,同时还对其它实施例进行了实验,均取得了相同或相似的疗效,这里不再一一列举。而且在实验中未发现不良反应、药物刺激,表明用药安全,疗效稳定可靠。
综上所述,本发明与现有技术相比,具有以下突出的有益技术效果:
1.本发明所制备的苦瓜多肽分子量较低,在5-6kDa之间,相较于传统苦瓜多肽分子量9-11kDa要小的多,更易于舌下吸收,提高药物利用率和疗效。
2.本发明采用的米曲霉作为菌种,采用米曲霉发酵后的苦瓜籽物料有利于苦瓜多肽的溶出,大大提高了多肽溶出率,得率高达15%左右,远高于现有方法的6.52%。
3.本发明苦瓜多肽的制备工艺与传统工艺相比,减少了丙酮脱脂工序,可降低由于丙酮处理过程中造成的苦瓜多肽复溶率低的问题,同时减少了有机溶剂用量,复溶率高达98%以上,远远高于现有技术的52.4%。
4.本发明所制备的低分子苦瓜多肽,口感较好无油腻感,且调节血糖能力强,非常有利于对糖尿病的治疗,是进一步提高糖尿病的治疗效果,有实际的应用价值和商业开发价值,是治疗糖尿病药物上的一大创新。
Claims (4)
1.一种治疗糖尿病的低分子量苦瓜多肽含片的制备方法,其特征在于,包括以下步骤:
1)苦瓜籽预处理
选取新鲜的苦瓜籽或无菌水清洗后浸泡6h的干燥苦瓜籽50kg,碾碎成颗粒,颗粒大小为0.5-1.5mm,进行预处理;
2)发酵苦瓜籽的制备
a)菌种活化:将米曲霉菌种接种于经过无菌验证的土豆培养基的试管斜面或平板中,土豆培养基是,马铃薯200g、葡萄糖20g、琼脂 15~20g,加蒸馏水1000mL制成,然后将接种后的斜面或平板在26~30℃下培养72h,得活化米曲霉菌种;
b)菌种扩培:取500mL的锥形瓶,添加液体培养基200mL,每升液体培养基中含有苦瓜籽粉10-30g、蛋白胨50g、苦瓜籽油2-8mL、硝酸钠2g、磷酸氢二钾1g、氯化钠0.5g和硫酸亚铁0.01g,将液体培养基进行湿热灭菌,灭菌温度121℃,时间为15min;培养基温度降到23-27℃,移至超净工作台,接入活化米曲霉菌种,在26-30℃恒温摇床中培养,摇床振荡频率为200r/min,培养时间为36h,得扩培菌种;
c)苦瓜籽发酵:苦瓜籽料与扩培菌种的质量体积比为18-22︰1 g/mL,质量体积是指固体质量以g计,液体以mL计,进行发酵,温度28-32℃,湿度85-95%,发酵48-72h,发酵期间每8h翻拌一次,再将发酵料干燥,粉碎,得干燥的发酵苦瓜籽粉;
3)乙醇提取
加质量浓度83-87%的乙醇溶液250L,于20-24℃下浸泡提取30-36h,离心、过滤,滤液蒸馏回收乙醇,加100L水,用氨水调pH值至9.6-9.8,离心,得上清液;
4)多肽沉淀
将上清液用1N的盐酸调pH值至3.5,加入90-110L无水乙醇,于4℃下放置16小时,得沉淀;
5)干燥粉碎
沉淀加无水乙醇溶解脱洗2-3次,每次10-20min,回收乙醇,收集固体沉淀,于42-45℃进行真空干燥,粉碎,过 100 目筛,得分子量为5-6kDa的苦瓜多肽;
6)制片:首先,将木糖醇过100目筛,过18目筛网制粒,湿颗粒于 40℃以下干燥,干颗粒过 16 目筛整粒,称重,再按常规制片方法加入苦瓜多肽和其余辅料,混匀,确定片重,压制成片;
所述的其余辅料为微粉硅胶、香精和柠檬酸。
2.根据权利要求1所述的治疗糖尿病的低分子量苦瓜多肽含片的制备方法,其特征在于,包括以下步骤:
1)苦瓜籽预处理
选取新鲜的苦瓜籽50kg,碾碎成颗粒,颗粒大小为1.0mm,进行预处理;
2)发酵苦瓜籽的制备
a)菌种活化:将米曲霉菌种接种于经过无菌验证的土豆培养基的试管斜面或平板中,然后将接种后的斜面或平板在28℃下培养72h,得活化米曲霉菌种;
b)菌种扩培:取500mL的锥形瓶,添加液体培养基200mL,每升液体培养基中含有苦瓜籽粉20g、蛋白胨50g、苦瓜籽油5mL、硝酸钠2g、磷酸氢二钾1g、氯化钠0.5g和硫酸亚铁0.01g,将液体培养基进行湿热灭菌,灭菌温度121℃,时间为15min;培养基温度降到25℃,移至超净工作台,接入活化米曲霉菌种,在28℃恒温摇床中培养,摇床振荡频率为200r/min,培养时间为36h,得扩培菌种;
c)苦瓜籽发酵:苦瓜籽料与扩培菌种的质量体积比为20︰1 g/mL,进行发酵,温度28℃,湿度85%,发酵60h,发酵期间每8h翻拌一次,再将发酵料干燥,粉碎,得干燥的发酵苦瓜籽粉;
3)乙醇提取
加质量浓度85%的乙醇溶液250L,于22℃下浸泡提取34h,离心、过滤,滤液蒸馏回收乙醇,加100L水,用氨水调pH值至9.7,离心,得上清液;
4)多肽沉淀
将上清液用1N的盐酸调pH值至3.5,加入100L无水乙醇,于4℃下放置16小时,得沉淀;
5)干燥粉碎
沉淀加无水乙醇溶解脱洗2次,每次20min,回收乙醇,收集固体沉淀,于44℃进行真空干燥,粉碎,过 100 目筛,得苦瓜多肽;
6)制片:首先,将木糖醇过100目筛,过18目筛网制粒,湿颗粒于 40℃以下干燥,干颗粒过 16 目筛整粒,称重,再按常规制片方法加入苦瓜多肽和其余辅料,混匀,确定片重,压制成片。
3.根据权利要求1所述的治疗糖尿病的低分子量苦瓜多肽含片的制备方法,其特征在于,包括以下步骤:
1)苦瓜籽预处理
选取新鲜的苦瓜籽50kg,碾碎成颗粒,颗粒大小为1.5mm,进行预处理;
2)发酵苦瓜籽的制备
a)菌种活化:将米曲霉菌种接种于经过无菌验证的土豆培养基的试管斜面或平板中,然后将接种后的斜面或平板在28℃下培养72h,得活化米曲霉菌种;
b)菌种扩培:取500mL的锥形瓶,添加液体培养基200mL,每升液体培养基中含有苦瓜籽粉20g、蛋白胨50g、苦瓜籽油5mL、硝酸钠2g、磷酸氢二钾1g、氯化钠0.5g和硫酸亚铁0.01g,将液体培养基进行湿热灭菌,灭菌温度121℃,时间为15min;培养基温度降到23-27℃,移至超净工作台,接入活化米曲霉菌种,在27℃恒温摇床中培养,摇床振荡频率为200r/min,培养时间为36h,得扩培菌种;
c)苦瓜籽发酵:苦瓜籽料与扩培菌种的质量体积比为20︰1 g/mL,质量体积是指固体质量以g计,液体以mL计,进行发酵,温度28℃,湿度85%,发酵72h,发酵期间每8h翻拌一次,再将发酵料干燥,粉碎,得干燥的发酵苦瓜籽粉;
3)乙醇提取
加质量浓度87%的乙醇溶液250L,于24℃下浸泡提取30h,离心、过滤,滤液蒸馏回收乙醇,加100L水,用氨水调pH值至9.8,离心,得上清液;
4)多肽沉淀
将上清液用1N的盐酸调pH值至3.5,加入90L无水乙醇,于4℃下放置16小时,得沉淀;
5)干燥粉碎
沉淀加无水乙醇溶解脱洗3次,每次10min,回收乙醇,收集固体沉淀,于45℃进行真空干燥,粉碎,过 100 目筛,得苦瓜多肽;
6)制片:首先,将木糖醇过100目筛,过18目筛网制粒,湿颗粒于 40℃以下干燥,干颗粒过 16 目筛整粒,称重,再按常规制片方法加入苦瓜多肽和其余辅料,混匀,确定片重,压制成片。
4.根据权利要求1所述的治疗糖尿病的低分子量苦瓜多肽含片的制备方法,其特征在于,包括以下步骤:
1)苦瓜籽预处理
选取无菌水清洗后浸泡6h的干燥苦瓜籽50kg,碾碎成颗粒,颗粒大小为0.5mm,进行预处理;
2)发酵苦瓜籽的制备
a)菌种活化:将米曲霉菌种接种于经过无菌验证的土豆培养基的试管斜面或平板中,然后将接种后的斜面或平板在28℃下培养72h,得活化米曲霉菌种;
b)菌种扩培:取500mL的锥形瓶,添加液体培养基200mL,每升液体培养基中含有苦瓜籽粉20g、蛋白胨50g、苦瓜籽油5mL、硝酸钠2g、磷酸氢二钾1g、氯化钠0.5g和硫酸亚铁0.01g,将液体培养基进行湿热灭菌,灭菌温度121℃,时间为15min;培养基温度降到25℃,移至超净工作台,接入活化米曲霉菌种,在30℃恒温摇床中培养,摇床振荡频率为200r/min,培养时间为36h,得扩培菌种;
c)苦瓜籽发酵:苦瓜籽料与扩培菌种的质量体积比为20︰1 g/mL,进行发酵,温度28℃,湿度90%,发酵48h,发酵期间每8h翻拌一次,再将发酵料干燥,粉碎,得干燥的发酵苦瓜籽粉;
3)乙醇提取
加质量浓度83%的乙醇溶液250L,于20℃下浸泡提取36h,离心、过滤,滤液蒸馏回收乙醇,加100L水,用氨水调pH值至9.6,离心,得上清液;
4)多肽沉淀
将上清液用1N的盐酸调pH值至3.5,加入110L无水乙醇,于4℃下放置16小时,得沉淀;
5)干燥粉碎
沉淀加无水乙醇溶解脱洗2次,每次15min,回收乙醇,收集固体沉淀,于42℃进行真空干燥,粉碎,过 100 目筛,得苦瓜多肽;
6)制片:首先,将木糖醇过100目筛,过18目筛网制粒,湿颗粒于 40℃以下干燥,干颗粒过 16 目筛整粒,称重,再按常规制片方法加入苦瓜多肽和其余辅料,混匀,确定片重,压制成片。
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