CN1072865A - 注射器 - Google Patents

注射器 Download PDF

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Publication number
CN1072865A
CN1072865A CN92114172A CN92114172A CN1072865A CN 1072865 A CN1072865 A CN 1072865A CN 92114172 A CN92114172 A CN 92114172A CN 92114172 A CN92114172 A CN 92114172A CN 1072865 A CN1072865 A CN 1072865A
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yeast
protease
lactase
extracting method
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瑟杰·M·马苏里克
安德瑞·N·梭克罗夫
马拉特·M·卡须里克夫
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M5/00—Devices for bringing media into the body in a subcutaneous, intra-vascular or intramuscular way; Accessories therefor, e.g. filling or cleaning devices, arm-rests
    • A61M5/178—Syringes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M5/00—Devices for bringing media into the body in a subcutaneous, intra-vascular or intramuscular way; Accessories therefor, e.g. filling or cleaning devices, arm-rests
    • A61M5/178—Syringes
    • A61M5/31—Details
    • A61M5/315—Pistons; Piston-rods; Guiding, blocking or restricting the movement of the rod or piston; Appliances on the rod for facilitating dosing ; Dosing mechanisms
    • A61M5/31596—Pistons; Piston-rods; Guiding, blocking or restricting the movement of the rod or piston; Appliances on the rod for facilitating dosing ; Dosing mechanisms comprising means for injection of two or more media, e.g. by mixing
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M5/00—Devices for bringing media into the body in a subcutaneous, intra-vascular or intramuscular way; Accessories therefor, e.g. filling or cleaning devices, arm-rests
    • A61M5/178—Syringes
    • A61M5/31—Details
    • A61M5/315—Pistons; Piston-rods; Guiding, blocking or restricting the movement of the rod or piston; Appliances on the rod for facilitating dosing ; Dosing mechanisms
    • A61M5/31511—Piston or piston-rod constructions, e.g. connection of piston with piston-rod
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M5/00—Devices for bringing media into the body in a subcutaneous, intra-vascular or intramuscular way; Accessories therefor, e.g. filling or cleaning devices, arm-rests
    • A61M5/178—Syringes
    • A61M5/31—Details
    • A61M5/315—Pistons; Piston-rods; Guiding, blocking or restricting the movement of the rod or piston; Appliances on the rod for facilitating dosing ; Dosing mechanisms
    • A61M5/31596—Pistons; Piston-rods; Guiding, blocking or restricting the movement of the rod or piston; Appliances on the rod for facilitating dosing ; Dosing mechanisms comprising means for injection of two or more media, e.g. by mixing
    • A61M2005/31598—Pistons; Piston-rods; Guiding, blocking or restricting the movement of the rod or piston; Appliances on the rod for facilitating dosing ; Dosing mechanisms comprising means for injection of two or more media, e.g. by mixing having multiple telescopically sliding coaxial pistons encompassing volumes for components to be mixed
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M5/00—Devices for bringing media into the body in a subcutaneous, intra-vascular or intramuscular way; Accessories therefor, e.g. filling or cleaning devices, arm-rests
    • A61M5/178—Syringes
    • A61M5/31—Details
    • A61M5/315—Pistons; Piston-rods; Guiding, blocking or restricting the movement of the rod or piston; Appliances on the rod for facilitating dosing ; Dosing mechanisms
    • A61M5/31525—Dosing
    • A61M5/31531—Microsyringes, e.g. having piston bore diameter close or equal to needle shaft diameter

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  • Health & Medical Sciences (AREA)
  • Vascular Medicine (AREA)
  • Engineering & Computer Science (AREA)
  • Anesthesiology (AREA)
  • Biomedical Technology (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Hematology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Infusion, Injection, And Reservoir Apparatuses (AREA)
  • Enzymes And Modification Thereof (AREA)

Abstract

尤其适用于医药目的注射器包含一注射筒体 (1)、一位于注射筒体(1)内而可在其中移动的活塞 (10)和一注射针(6)，为确保可抽取精确量的活性成 分和稀释液，活塞(10)包含至少二可沿注射筒体(1) 的纵轴相对移动的活塞元件(2、3)。

Description

本发明为一种酵母乳糖酶的提取方法，该方法属于化学溶剂破壁提取的范围，适用于对酵母膜酶的提取和初步纯化。
乳糖酶广泛用于乳制品工业，在奶酪和低乳糖牛奶的加工过程中加入少量乳糖酶可加快乳糖水解速度，提高产品质量。此酶亦用于成人和婴幼儿先天性或继发性乳糖不耐受症的治疗。乳糖酶是某些酵母的胞内酶，固定在酵母细胞膜的内侧，属于膜酶。常规的提取方法是将含乳糖酶的酵母从培养液中收获、榨干，用物理或化学的方法破细胞壁，然后提取。如匀桨法，机械研磨法，甲苯或乙酸乙酯等有机溶剂法等（Y.Ito.et.al：Eur.J.Biochem.105  85-92  1980）但是，用这些方法处理时间较长，乳糖酶的释放包括酶从膜结构上游离的过程，此时，胞内其他物质也释放出来，其中包括蛋白酶类。蛋白酶很难从酶制品中除去，少量蛋白酶的存在可使乳糖酶在较短期内大量失活。因此，应缩短乳糖酶提取时间并降低已释放的蛋白酶活性，从而提高乳糖酶的保存性能。
本发明提出一种改进的酵母乳糖酶提取方法，可在较短时间内获得较高的回收率，并选择性地降低酵母蛋白酶的活性，提高乳糖酶的保存性能。
本方案采用多种无毒或低毒有机溶剂按一定比例配制成破膜溶剂，使酵母细胞在较短时间内破膜，并使用外源性蛋白酶加速乳糖酶从酵母细胞膜上释放，亦部分破坏了已释放的酵母蛋白酶，再针对不同的蛋白酶使用不同的蛋白酶抑制物，使之灭活。达到初步纯化之目的。这一过程，可使酵母本身的蛋白酶活性大部分被消除，缩短了提取时间，提高了提取百分率，同时又改善了乳糖酶的保存性能。
此方法还适用于对酵母蔗糖酶，酵母麦芽糖酶及其它酵母膜酶的提取。
本发明提出的破膜溶剂为醇类、酯类与氯仿组成的混合溶剂。其中醇类为2-4个碳原子的醇，占混合溶剂的50-80%（V/V）;酯类为甲酸、乙酸或丁酸的C2-C5碳醇酯，占15-40%（V/V）;氯仿占5-10%（V/V）。采用的外源性蛋白酶是选取对乳糖酶从膜上游离释放有效，对乳糖酶损伤小，并便于从乳糖酶提取液中除去的蛋白酶。如：胰蛋白酶、糜蛋白酶、木瓜蛋白酶、枯草杆菌蛋白酶、地衣芽胞杆菌蛋白酶、米曲蛋白酶。黑曲蛋白酶等均有不同的效果。针对不同蛋白酶选用大豆胰蛋白酶抑制因子、碘乙酸（或碘乙酰胺）等作为蛋白酶抑制物，以去除乳糖酶提取液中的外源性蛋白酶。
本发明具体方案可见于以下实施例。
实施例一：100克糊状湿酵母，加100ml破膜溶剂，破膜溶剂配方为：乙醇60ml、乙酸乙酯30ml、氯仿10ml。搅拌10-30分钟，使酵母破膜。得到破膜混悬液约为180ml，加入PH6-10的含适量木瓜蛋白酶的缓冲液至1800ml，室温搅拌6-24小时，使乳糖酶从破碎的细胞壁上游离溶解，然后用滴定法加入碘乙酸（或碘乙酰胺）适量，使木瓜蛋白酶被抑制。4000-7000rpm离心12分钟去除沉淀，上清液即为初步纯化的乳糖酶提取液。提取液中几乎无残余的蛋白酶活性，有良好的保存性能。
实施例二：100克糊状湿酵母，加100ml破膜溶剂，破膜溶剂配方为：异丙醇50-80%，甲酸乙酯15-40%，氯仿5-10%，搅拌10-30分钟使酵母破膜，加入PH6-10的磷酸盐缓冲液至1800ml，内含适量胰蛋白酶，搅拌6-24小时，使乳糖酶从破碎的细胞壁上游离、溶解，然后滴加大豆胰蛋白酶抑制因子适量，使胰蛋白酶正好被抑制，4000-7000rpm离心12分钟去除沉淀，上清液即为提纯的乳糖酶提取液。经测定，提取液中几乎无蛋白酶活性，乳糖酶保存性能良好。
实施例三：100克糊状湿酵母，加100ml破膜溶剂，破膜溶剂配比为：乙醇60ml，乙酸乙酯30ml，氯仿10ml，搅拌处理10-30分钟，加入PH6-10的磷酸盐缓冲液，其中不含外源性蛋白酶，加至1800ml，搅拌24-48小时，使乳糖酶自细胞膜上游离，4000-7000rpm离心12分钟，去除沉淀，得乳糖酶提取液。
与例一、例二相比，由于例三中未使用外源性蛋白酶处理破膜后的酵母碎片，乳糖酶从细胞膜碎片上游离较慢，搅拌处理时间需增加2-4倍，同时，乳糖酶的提取率较例一、例二为低，保存性能也差。
实施例四，100克糊状湿酵母，加100ml破膜溶剂，破膜溶剂配比为：乙醇50-80%，丁酸丁酯15-40%，氯仿5-10%，搅拌处理10-30分钟，加入9倍于处理液量的，PH6-10的缓冲液，内含适量外源性蛋白酶（黑曲蛋白酶或米曲蛋白酶或地衣芽胞杆菌蛋白酶或糜蛋白酶任取一种），搅拌6-24小时后，用滴定法加入大豆胰蛋白酶抑制因子适量，使外源性蛋白酶被抑制，4000-7000rpm离心12分钟，滤去沉淀，得初步纯化的乳糖酶提取液。该提取液中乳糖酶活性良好，回收率理想，且保存性能好。

Claims (5)

1、一种酵母乳糖酶的提取方法，属于用化学溶剂破壁处理的范围，其特征在于：用醇类(C2-C4的醇)、酯类(甲酸、乙酸或丁酸的C2-C5碳醇酯)和氯仿组成的混合溶液处理糊状湿酵母，搅拌10-30分钟，使之破壁液化，每份此混合物中加入PH6-10的缓冲液9份(V/V)，搅拌24-48小时，离心法除去沉淀物，得到含酵母乳糖酶的提取液。
2、根据权利要求1所述酵母乳糖酶的提取方法，其特征在于：所述PH6-10的缓冲液中加入适量的外源性蛋白酶，搅拌6-24小时后用滴定法加入蛋白酶抑制物，然后离心除去沉淀物，得到含酵母乳糖酶的提取液。
3、根据权利要求1或2所述酵母乳糖酶的提取方法，其特征在于：所述外源性蛋白酶为木瓜蛋白酶，或黑曲蛋白酶，或米曲蛋白酶，或地衣芽胞杆菌蛋白酶，或糜蛋白酶，或胰蛋白酶;所述蛋白酶抑制物是大豆胰蛋白酶抑制因子，或碘乙酸，或碘乙酰胺。
4、根据权利要求1所述酵母乳糖酶的提取方法，其特征在于：所述混合溶液的配比（V/V）为：醇类50-80%，酯类15-40%，氯仿5-10%。
5、根据权利要求1或2所述酵母乳糖酶的提取方法，其特征在于：此法还适用于对酵母蔗糖酶、酵母麦芽糖酶及其它酵母膜酶的提取。
CN92114172A 1991-11-29 1992-11-28 注射器 Pending CN1072865A (zh)

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SU5015271 1991-11-29
SU5015271 1991-11-29

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US (1) US5354285A (zh)
EP (1) EP0545324A1 (zh)
JP (1) JPH05237195A (zh)
KR (1) KR930009615A (zh)
CN (1) CN1072865A (zh)
AU (1) AU2969192A (zh)
BR (1) BR9204597A (zh)
CA (1) CA2063840A1 (zh)
DE (1) DE4206300A1 (zh)
FI (1) FI925420A (zh)
IL (1) IL101360A0 (zh)
MX (1) MX9201683A (zh)
NO (1) NO924586L (zh)
ZA (1) ZA929225B (zh)

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MX9201683A (es) 1993-05-01
ZA929225B (en) 1993-05-26
NO924586D0 (no) 1992-11-27
KR930009615A (ko) 1993-06-21
DE4206300A1 (de) 1993-06-03
NO924586L (no) 1993-06-01
FI925420A0 (fi) 1992-11-27
FI925420A (fi) 1993-05-30
CA2063840A1 (en) 1993-05-30
BR9204597A (pt) 1993-06-01
JPH05237195A (ja) 1993-09-17
US5354285A (en) 1994-10-11
IL101360A0 (en) 1992-11-15
EP0545324A1 (en) 1993-06-09
AU2969192A (en) 1993-06-03

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