CN100465285C - 肌肽的提取方法 - Google Patents
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Abstract
本发明公开了一种肌肽的提取方法，该方法依次由下述过程组成：将动物肉去除非肌肉组织物并洗净、沥干；磨细加水搅匀成浆液；进行双酶水解，然后进行酶灭活；分离出清液；用羟基磷灰石层析柱进行吸附，用超滤膜进行超滤，收集滤出液，对肌肽原液进行喷雾干燥，收集干燥粉，真空包装。这种提取方法不但安全、无污染，而且原料易得、收率高，生产成本低，适合大规模工业化生产。
Description
技术领域
本发明涉及医药化工的生产工艺，具体地说是从动物骨骼肌中提取肌肽的方法。
背景技术
肌肽是一种天然的二肽，存在于肌肉中，由β-丙氨酸和组氨酸组成。Boldyrev(1988)等报道肌肽具有抗氧化作用，可以抑制由铁、铜、肌红蛋白和脂肪氧化酶引起的脂肪氧化反应。肌肽具有比BHA、BHT、TBHQ、PG及α-生育酚更强的抗氧化作用。Decker等(1990)也研究发现肌肽可以抑制体内由铁、血红素蛋白、脂肪氧化酶和单质氧催化的氧化反应。肌肽还可以抑制酸败气味的形成和颜色的变化，加入肌肽的样品在贮藏1个月后，其酸败气味显著低于加入BHT、α-生育酚和三聚磷酸钠的样品，且它的护色效果比BHT、α-生育酚和三聚磷酸钠要好，BHT、α-生育酚和三聚磷酸钠对保护颜色是无效的(Decker等，1991；1995)。加入肌肽的虾类在贮藏45天后，其虾壳颜色、口味等基本无变化。目前肌肽广泛应用于药物辅剂、食品添加剂、化妆品及饲料添加剂等领域，是一种高效低毒的天然抗氧化剂、护色剂。
现有的肌肽的制备方法为利用化学合成方法而得(F.J.Vinicket al.，J.Org.Chem.1983，48(3)，392等)，其缺点是使用大量的有机化学溶剂：联氨化合物、硫化物等是剧毒的化学物质，叠氮化合物为易燃易爆化学品，必须在防爆车间内生产，操作麻烦，污染环境，损害人体健康；而且设备投资大，生产效率低，生产周期长，生产成本高。
发明内容
针对现有的肌肽制备方法存在的危害及生产效率低、生产成本高的问题，本发明目的在于提供一种从动物骨骼肌中提取肌肽的生产工艺。
本发明采用如下技术解决方案：
(1)取新鲜或冻存健康动物肉，去除非肌肉组织物，用注射用水洗净，沥干；
(2)先用绞肉机绞成碎块，用胶体磨磨细，收集匀浆液，加入注射用水搅匀成浆液；
(3)将浆液加温至42℃～45℃，用酸调pH至1.5～1.7，加入胃蛋白酶，或酸性蛋白酶或木瓜酶，搅匀后，维持pH1.5～1.7、42℃～45℃的条件水解；
(4)用碱将水解后的浆液调pH至7.5～7.7，然后加入胰酶或碱性蛋白酶搅匀后，维持在pH7.5～7.7、42℃～45℃水解，酶解结束后加热升温至100℃，保温5～15分钟进行酶灭活，降温至30℃以下；
(5)用酸调pH至2.8～3.0，静置过夜；
(6)进行固液分离，分离出的清液用碱调pH至6.8～7.0，备用；
(7)用注射用水将羟基磷灰石调成糊状装柱，装柱后用pH5.8～6.8的酸性磷酸盐缓冲液清洗，再用pH5.8～6.8的酸性磷酸盐缓冲液平衡，将上述滤出液上羟基磷灰石层析柱进行吸附，用pH5.8～6.8的酸性磷酸盐缓冲液洗脱；
(8)收集目标物，用滤膜进行超滤，收集滤出液，对肌肽原液进行喷雾干燥，收集干燥粉；
(9)取样进行微生物限度、含量测定、酸度检测。
所述的动物肉可以为牛肉、兔肉、鸡肉、鱼肉、羊肉或猪肉等。
所述的调pH值用的碱可以为氢氧化钠、氢氧化钙、氢氧化钾、碳酸氢钠或氨水等。
所述的调pH值用的酸可以为盐酸、磷酸、柠檬酸、硫酸等。
用酶进行水解的过程，可以通过控制水解时间来掌握水解进程，水解时间短，水解不充分造成收率低，但水解时间过长又会延长生产周期，增加生产成本。一般步骤(3)中水解时间可以为420～540分钟。同样，所述的步骤(4)中水解时间为120～240分钟。
所述的酸性磷酸盐缓冲液可以为10～30mmol/L。
为保证干燥效果，所述的离心喷雾干燥器进料温度可以设为45℃，进出口温度可以为140℃～70℃，喷雾器转速8000r/min。
所述的超滤可以采用截留分子量为3～8KD的超滤膜。
本发明通过对动物骨骼肌进行双酶水解，将大分子蛋白质水解成小分子多肽，然后使用羟基磷灰石层析柱吸附分离纯化，可以分段收集得到单一、纯度比较高的肌肽。这种方法与合成方法相比，具有如下优点：
1、不使用联氨化合物、硫化物、叠氮化合物等剧毒、易燃易爆化学品，新鲜或冻存动物肉直接经双酶酶解等生产工艺提取肌肽，具安全无污染、少投入生产设备、减少生产环节等优点。
2、双酶水解肌肉组织匀浆液，采用抽取、残渣离心获得上清液，经过滤、上羟基磷灰石层析柱吸附分离纯化，超滤等生产工艺，水解完全，可除去杂蛋白和大分子杂质，达到很好的分离效果。
3、采用双酶酶解，抽取、残渣离心获得上清液，经过滤、上羟基磷灰石层析柱吸附分离纯化，超滤等生产工艺，优点是生产步骤、周期大大缩短、收率提高、生产成本降低、生产环境要求降低、操作简便，减少了药品受微生物污染、变质的机会。
4、采用离心喷雾干燥，可以得到淡黄色粉状颗粒，颗粒均匀，无褐色杂质溶解性好。
5、制备工艺简单易行，原料易得，反应条件温和，收率高，稳定性好，而且周期短，对制备设备及场所要求也很低，适合于大规模工业化生产。
具体实施方式
下面结合具体实施例对本发明的肌肽的提取方法作进一步说明。
实施例：
取经检验合格的新鲜或冻存健康牛肉100.0kg，去除结缔组织、油脂、碎骨等非肌肉组织物，用注射用水洗净，沥干，称重。先用绞肉机绞成碎块，用调节刻度至200目的胶体磨磨细，收集匀浆液，加入150.0kg(1.5倍重)的注射用水搅匀。将浆液加温至42℃～45℃，用4.5％盐酸调pH至1.5～1.7，按每kg浆液加入10万国际单位(比活力)胃蛋白酶的比例，加入2500万国际单位(比活力)胃蛋白酶，搅匀后，维持pH1.5～1.7、42℃～45℃的条件水解8小时。用18％ NaOH将胃蛋白酶水解浆液调pH至7.5～7.7，按每kg浆液加入4万国际单位(比活力)胰酶的比例，加入1000万国际单位(比活力)胰酶，搅匀后，维持在pH7.5～7.7、42℃～45℃水解4小时，酶解结束后加热升温至100℃，保温15分钟进行酶灭活，降温至30℃以下。用4.5％盐酸调pH2.8～3.0，静置过夜。抽滤得上清液176.0kg，残渣用离心机离心，收集上清液得53.0kg，合并上清液，共229.0kg，进行粗过滤，收集滤出液得227.0kg，用18％NaOH调pH至6.8～7.0，备用。用注射用水将羟基磷灰石调成糊状，采用淤浆式法装柱，装柱压力0.5Mpa。装柱后先用400mmol/L pH6.8磷酸盐缓冲液清洗，再用20mmol/L pH6.8磷酸盐缓冲液平衡，将上述滤出液上羟基磷灰石层析柱进行吸附，用20mmol/L pH6.8磷酸盐缓冲液洗脱。收集目标物得285.0kg，用对分子截留为3K的滤膜进行超滤，收集滤出液得274.0kg，将离心喷雾干燥器进料温度设为45℃，进出口温度为140℃～70℃，喷雾器转速8000r/min，对肌肽原液进行喷雾干燥，收集干燥粉，得肌肽粉26.38kg。真空包装，备用。取样进行微生物限度、含量测定、酸度等检测。
六批中试结果：
| 批号 | 20060801 | 20060802 | 20060803 | 20060804 | 20060805 | 20060806 |
| 骨骼肌 | 牛肉 | 羊肉 | 猪肉 | 鱼肉 | 鸡肉 | 兔肉 |
| 投料量 | 100.0kg | 100.0kg | 100.0kg | 100.0kg | 100.0kg | 100.0kg |
| 肌肽粉 | 26.3kg | 24.6kg | 25.8kg | 22.5kg | 26.1kg | 25.7kg |
| 性状 | 淡黄色粉末 | 淡黄色粉末 | 淡黄色粉末 | 淡黄色粉末 | 淡黄色粉末 | 淡黄色粉末 |
| 鉴别试验 | 符合规定 | 符合规定 | 符合规定 | 符合规定 | 符合规定 | 符合规定 |
| 干燥失重(％) | 0.53 | 0.65 | 0.58 | 0.68 | 0.64 | 0.70 |
| pH值 | 6.24 | 6.28 | 6.17 | 6.23 | 6.18 | 6.10 |
| 微生物限定 | 符合规定 | 符合规定 | 符合规定 | 符合规定 | 符合规定 | 符合规定 |
| 多肽含量(mg/g) | 94.79 | 93.90 | 94.00 | 94.86 | 93.87 | 94.61 |
多肽含量根据国家药品标准WS-10001-(HD-0874)-2002中脑苷肌肽注射液多肽含量的方法测定。
Claims (10)
1、一种肌肽的提取方法，该方法依次由下述过程组成：
(1)取新鲜或冻存健康动物肉，去除非肌肉组织物，用注射用水洗净，沥干；
(2)先用绞肉机绞成碎块，用胶体磨磨细，收集匀浆液，加入注射用水搅匀成浆液；
(3)将浆液加温至42℃～45℃，用酸调pH至1.5～1.7，加入胃蛋白酶，或酸性蛋白酶或木瓜酶，搅匀后，维持pH1.5～1.7、42℃～45℃的条件水解；
(4)用碱将水解后的浆液调pH至7.5～7.7，然后加入胰酶或碱性蛋白酶搅匀后，维持在pH7.5～7.7、42℃～45℃水解，酶解结束后加热升温至100℃，保温5～15分钟进行酶灭活，降温至30℃以下；
(5)用酸调pH至2.8～3.0，静置过夜；
(6)进行固液分离，分离出的清液用碱调pH至6.8～7.0，备用；
(7)用注射用水将羟基磷灰石调成糊状装柱，装柱后用pH5.8～6.8的酸性磷酸盐缓冲液清洗，再用pH5.8～6.8的酸性磷酸盐缓冲液平衡，将上述滤出液上羟基磷灰石层析柱进行吸附，用pH5.8～6.8的酸性磷酸盐缓冲液洗脱；
(8)收集目标物，用滤膜进行超滤，收集滤出液，对肌肽原液进行喷雾干燥，收集干燥粉；
(9)取样进行微生物限度、含量测定、酸度检测。
2、如权利要求1所述的肌肽的提取方法，其特征在于：所述的动物肉为牛肉、兔肉、鸡肉、鱼肉、羊肉或猪肉。
3、如权利要求1所述的肌肽的提取方法，其特征在于：所述的调pH值用的碱为氢氧化钠、氢氧化钙、氢氧化钾、碳酸氢钠或氨水。
4、如权利要求1所述的肌肽的提取方法，其特征在于：所述的调pH值用的酸为盐酸、磷酸、柠檬酸或硫酸。
5、如权利要求1所述的肌肽的提取方法，其特征在于：所述的步骤(3)中水解时间为420～540分钟。
6、如权利要求1所述的肌肽的提取方法，其特征在于：所述的步骤(4)中水解时间为120～240分钟。
7、如权利要求1所述的肌肽的提取方法，其特征在于：在所述的步骤(6)中，先抽滤得上清液，残渣用离心机离心，收集上清液，合并上清液，进行过滤，分离出清液。
8、如权利要求1所述的肌肽的提取方法，其特征在于：所述的磷酸盐缓冲液为10～30mmol/L。
9、如权利要求1所述的肌肽的提取方法，其特征在于：所述的离心喷雾干燥器进料温度设为45℃，进出口温度为140℃～70℃，喷雾器转速8000r/min。
10、如权利要求1所述的肌肽的提取方法，其特征在于：所述的超滤采用截留分子量为3～8KD的超滤膜。
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| CN106366159A (zh) * | 2016-08-31 | 2017-02-01 | 精晶药业股份有限公司 | 一种肌肽的提取纯化方法 |
| CN106366158A (zh) * | 2016-08-31 | 2017-02-01 | 精晶药业股份有限公司 | 一种肌肽的结晶方法 |
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| CN103621761A (zh) * | 2012-08-24 | 2014-03-12 | 天津科技大学 | 一种减肥肽的制备 |
| CN104232715A (zh) * | 2014-08-20 | 2014-12-24 | 青岛贝尔特生物科技有限公司 | 一种蛋白低聚肽的制备方法 |
| CN105124319A (zh) * | 2015-09-30 | 2015-12-09 | 江苏省协同医药生物工程有限责任公司 | 犬粮肉味诱食剂的制备方法 |
| CN108157580A (zh) * | 2017-12-29 | 2018-06-15 | 浙江大飞龙动物保健品股份有限公司 | 一种猪肉功能性多肽及其制备方法 |
| CN110150449A (zh) * | 2018-01-17 | 2019-08-23 | 浙江大飞龙动物保健品股份有限公司 | 猪肉功能性多肽的用途 |
| CN110419618A (zh) * | 2019-07-25 | 2019-11-08 | 李猷 | 一种富含肌肽的动物蛋白粉及其制备方法 |
| CN113332414B (zh) * | 2021-05-31 | 2022-02-11 | 北京四环制药有限公司 | 一种稳定性好的脑苷肌肽注射液及其制备方法和其应用 |
| CN114836491B (zh) * | 2022-05-30 | 2023-03-14 | 广州恒雅生物化工有限公司 | 一种抗老亮肤的肌肽制备方法及其在化妆品的应用 |
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| JP2001029042A (ja) * | 1999-07-19 | 2001-02-06 | Nikken Foods Co Ltd | 食用天然カルノシン含有組成物 |
| CN1532205A (zh) * | 2003-03-19 | 2004-09-29 | 四川三高生化股份有限公司 | 一种肌肽的生产方法 |
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| CN1532205A (zh) * | 2003-03-19 | 2004-09-29 | 四川三高生化股份有限公司 | 一种肌肽的生产方法 |
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| CN106366159A (zh) * | 2016-08-31 | 2017-02-01 | 精晶药业股份有限公司 | 一种肌肽的提取纯化方法 |
| CN106366158A (zh) * | 2016-08-31 | 2017-02-01 | 精晶药业股份有限公司 | 一种肌肽的结晶方法 |
| CN106366158B (zh) * | 2016-08-31 | 2019-11-05 | 精晶药业股份有限公司 | 一种肌肽的结晶方法 |
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