WO2006114039A1 - Procede de separation de nisine d'un liquide de fermentation de galactococcus lactique - Google Patents
Procede de separation de nisine d'un liquide de fermentation de galactococcus lactique Download PDFInfo
- Publication number
- WO2006114039A1 WO2006114039A1 PCT/CN2006/000638 CN2006000638W WO2006114039A1 WO 2006114039 A1 WO2006114039 A1 WO 2006114039A1 CN 2006000638 W CN2006000638 W CN 2006000638W WO 2006114039 A1 WO2006114039 A1 WO 2006114039A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- nisin
- membrane
- separating
- fermentation broth
- molecular weight
- Prior art date
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/315—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci
Definitions
- the present invention relates to a method for separating a microorganism product, and more particularly to a method for separating a lactic acid streptomycin by concentrating a fermentation broth of Lactococcus lactis.
- Nisin is a small molecule peptide produced by certain Lactococcus Lactis with a molecular weight of approximately 3510 D a. This bacterium has a strong inhibitory effect on many Gram-positive bacteria, especially Bacillus, Clostridium, Staphylococcus, Listeria, etc., which cause food spoilage, and nisin can be digested by humans, so lactic acid As a food preservative, streptomycin has natural and non-toxic properties and is widely used as an antiseptic and preservative for dairy products, canned foods, meat products and beverages.
- the strain used was laccase-producing strain (Lactococus lactis subsp. lactis) Y-72, and the detection strain was Micrococcus flavus.
- NCIB 8166 was studied by the Chinese Academy of Sciences.
- the fermentation medium is: soy protein ⁇ 1. 0%, beef extract 0. 25%, yeast extract 0. 25%, Na 2 HP0 4 ⁇ 12H0 2 1. 5%, MgS0 4 ⁇ 7H 2 00. 012% , Nature PH value; nisin titer assay medium was: 0.8% tryptone, yeast extract, 0. 5%, NaClO 5%, NaHP0 4 ⁇ 12 00. 2%, agar 1.
- nisin standard produced by Aplin & Barrett LTD, tryptone: Oxoid LTD
- polyterpene produced in Japan, other medium is produced by Beijing Haidian District Microbial Culture Products Factory
- Growth assay measured at 600 nm with a Model 721 spectrophotometer
- Determination of streptavidin titer The agar diffusion method was used to measure the size of the inhibition zone on the test plate, and the nisin standard was used as a control to calculate the titer of the test solution according to the relevant formula.
- the mixture is heated at 70-100 ° C for 1-60 minutes, the bacteria are killed and the nisin is completely released in the fermentation broth;
- the adsorbent is adsorbed, and the mixture is stirred for 0.5 to 3 hours to prepare an adsorbent-nisin complex; the precipitate of the prepared adsorbent-nisin complex is washed twice with distilled water, and then 10% to 20% of the fermentation broth volume is used.
- the desorbent is desorbed, and the desorbent used is an aqueous solution of polyethylene glycol and an organic acid salt or a mineral acid salt, and is desorbed by stirring at 20 to 40 ° C for 5 to 60 minutes.
- the method requires adsorption, desorption, and addition of an adsorbent and a desorbent.
- the production process is complicated, the cost is high, the activity of the desired product is easily destroyed, the energy consumption is high, the activity is kept low, and the efficiency is low.
- the object of the present invention is to provide a method for separating and extracting nisin with simple production process, low cost, high yield, high activity and good separation effect.
- the present invention provides a method for separating nisin from a fermentation broth comprising releasing nisin, separation and spray drying, wherein the separation is greater than or less than a molecular weight by membrane filtration, respectively.
- the substance of the molecular weight of nisin is separated from the fermentation broth.
- the cerium is filtered with an inorganic membrane or a tubular membrane to remove substances having a molecular weight greater than that of nisin, such as cells and solids, from the fermentation broth.
- the inorganic membrane or tubular membrane has a pore diameter of from 1 to 600,000, preferably from 15 to 500,000; and the temperature is preferably from 15 to 45 °C, most preferably from 25 to 40 °C.
- a volumetric membrane is used to remove a substance having a molecular weight smaller than that of nisin from the fermentation broth, wherein the volume of the wound membrane is 0. 2 million.
- the temperature is preferably from 15 to 45 ° C, most preferably from 25 to 40. C.
- the ultra-filtered fermentation broth concentrate has a concentration of 100,000-500,000 IU /ml, and the filler solid salt is added to make the salt content of the spray-dried 10-50%, spray-drying on the spray dryer, hot air
- the inlet temperature is 120-180 ° C, and the outlet temperature is 70-100 ° C, which is a powdered food grade nisin product.
- Nisin is nisin.
- the inorganic or tubular membranes used in the present invention are made of metal oxides, ceramics, etc., and have a long life.
- the membrane main components are mainly supplied from abroad and assembled domestically. Manufacturers include Cerrer (France), T0K (Japan), Norton (USA), Mitsui (Japan), Nipongaishi (Japan), Sfec (France), Corning (USA), DDS (Denmark) and other companies.
- the roll film is made of organic materials, mainly including various materials such as polysulfone, polyethersulfone, polyacrylonitrile, polyvinylidene fluoride, cellulose acetate, etc.
- the main film parts are generally imported by foreign companies, and assembled and matched in China. The performance is superior. Manufacturers mainly include companies such as Pall (USA), Amicon (USA), and HAYWARD (USA).
- the ultra-filtered fermentation broth concentrate is added with filler solid salt to make the salt content after spray drying to 30%, spray-drying on a spray dryer, the hot air inlet temperature is 120 ° C, and the outlet temperature is 70 ° C, ie A powdered food grade nisin product.
- the inorganic membrane module is manufactured by Cerrer (France), and the membrane module is
- the specific steps for extracting nisin from the fermentation broth of Lactococcus lactis are as follows -
- the fermentation broth containing nisin produced by fermentation of 200000 ml of Lactococcus lactis is adjusted to pH 5.3 with concentrated sulfuric acid, and heated at 100 ° C for 1 minute to completely release nisin in the fermentation broth;
- the fermentation broth having a molecular weight greater than the molecular weight of nisin, such as cells and solids, is obtained by filtration through an inorganic membrane at a temperature of 20 ° C, and the membrane has a pore diameter of 150,000;
- the above-mentioned ultra-filtered fermentation broth concentrate is added with filler solid salt to make the salt content after spray drying to 40%, spray-drying on a spray dryer, the hot air inlet temperature is 150 ° C, and the outlet temperature is 80 ° C. That is, a powdered food grade nisin product.
- the inorganic membrane module was manufactured by T0K (Japan), and the wound membrane module was manufactured by Ainicon (USA).
- the specific steps for extracting nisin from the fermentation broth of Lactococcus lactis are as follows -
- the fermentation broth containing nisin produced by fermentation of 200000 ml of Lactococcus lactis is adjusted to pH 2.0 with concentrated hydrochloric acid, and heated at 80 ° C for 30 minutes to completely release nisin in the fermentation broth;
- the pressure of the operating pressure is 1. 5MPa pass Over-volume membrane ultrafiltration to remove substances with a molecular weight lower than that of nisin to obtain a concentration of 300,000 IU/ml fermentation broth, wherein the volume of the ultrafiltration membrane is 10,000; 4) Spray drying:
- the ultra-filtered fermentation broth concentrate is added with filler solid salt to make the salt content of the spray dried to 50%, spray-drying on a spray dryer, the hot air inlet temperature is 180 ° C, and the outlet temperature is 100 ° C, that is, A powdered food grade nisin product.
- the inorganic membrane module is manufactured by Norton (USA), and the membrane module is
- the fermentation broth containing nisin produced by fermentation of 200000 ml of Lactococcus lactis is adjusted to pH 2.5 with concentrated hydrochloric acid, and heated at 85 ° C for 20 minutes to completely release nisin in the fermentation broth;
- the fermentation broth is at a temperature of 30 ° C, and the operating pressure is 1. 2 MPa through a tubular membrane filtration to obtain a fermentation broth having a molecular weight greater than that of nisin, such as cells and solids, wherein the pore diameter of the membrane is 100,000. ;
- OMPa The O. Over-volume membrane ultrafiltration to remove substances with a molecular weight lower than that of nisin to obtain a fermentation broth concentrate at a concentration of 500,000 IU/ml, wherein the pore size of the wound membrane is 20,000; 4) Spray drying:
- the above-mentioned ultra-filtered fermentation broth concentrate is added with filler solid salt to make the salt content after spray drying to 10%, spray-drying on a spray dryer, the hot air inlet temperature is 160 ° C, and the outlet temperature is 90 ° C, that is, A powdered food grade nisin product.
- the inorganic membrane module was manufactured by Mitsui (Japan) and the wound membrane module was manufactured by Pall (USA).
- the fermentation broth having a molecular weight greater than that of nisin, such as a cell and a solid, is obtained by a tubular membrane filtration at a temperature of 55 ° C and an operating pressure of 0.5 MPa.
- the pore diameter of the membrane is 300,000. ;
- the above-mentioned ultrafiltered fermentation broth concentrate is added with a filler solid salt to make the salt content after spray drying 50%.
- Spray drying was carried out on a spray dryer with a hot air inlet temperature of 165 ° (:, an outlet temperature of 85 ° C, which gave a powdered food grade nisin product.
- the inorganic membrane module was manufactured by Nipongaishi (Japan), and the wound membrane module was manufactured by Ami con (USA).
- nisin - first take 200,000 ml of Lactococcus lactis fermentation-containing fermentation broth containing nisin with concentrated hydrochloric acid to adjust the pH to 3.5, heat at 100 ° C for 1 minute, so that nisin is completely released In the fermentation broth;
- the fermentation broth having a molecular weight greater than the molecular weight of nisin, such as cells and solids, is obtained by filtration through an inorganic membrane at a temperature of 10 ° C, and the membrane has a pore diameter of 450,000;
- the pressure of the fermentation broth is at a temperature of 30 ° C, the operating pressure is 1.
- the ultra-filtered fermentation broth concentrate is added to the filler solid salt to make the salt content of the spray dried to 20%, spray-drying on a spray dryer, the hot air inlet temperature is 150 ° C, and the outlet temperature is 80 ° C. That is, a powdered food grade nisin product.
- the inorganic membrane module is manufactured by DDS (Denmark) and the wound membrane module is manufactured by HAYWARD (USA).
- the specific steps for extracting nisin from the fermentation broth of Lactococcus lactis are as follows -
- the fermentation broth containing nisin produced by fermentation of 200000 ml of Lactococcus lactis is adjusted to pH 5.3 with concentrated sulfuric acid, and heated at 100 ° C for 1 minute to completely release nisin in the fermentation broth;
- the fermentation broth having a molecular weight greater than the molecular weight of nisin, such as cells and solids, is obtained by filtration through an inorganic membrane at a temperature of 10 ° C, and the operating pressure is 1. 6 MPa, wherein the pore diameter of the membrane is 500,000;
- the pressure of the operating pressure is 2. 5MPa pass Over-volume membrane ultrafiltration to remove substances with a molecular weight lower than that of nisin to obtain a fermentation broth concentrate at a concentration of 350,000 IU/ml, wherein the pore size of the wound membrane is 15,000; 4) Spray drying:
- the above-mentioned ultra-filtered fermentation broth concentrate is added to the filler solid salt to make the salt content after spray drying 45%, spray-drying on a spray dryer, the hot air inlet temperature is 150 ° C, and the outlet temperature is 80 ° C. That is, a powdered food grade nisin product.
- the inorganic membrane module was manufactured by Corning (USA) and the membrane module was manufactured by Amicon (USA).
- the method for separating and extracting nisin from the fermentation liquid provided by the invention has simple production process, greatly reduced cost, and no damage to the target product, and the activity remains high due to the use of inorganic membrane filtration and roll membrane ultrafiltration.
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Gastroenterology & Hepatology (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Separation Using Semi-Permeable Membranes (AREA)
Description
从发酵液中分离提取乳酸链球菌素的方法
技术领域
本发明涉及微生物产物的分离方法，具体讲涉及一种利用膜对乳 酸乳球菌发酵液进行浓缩， 分离乳酸链球菌素的方法。 技术背景
乳酸链球菌素是由某些乳酸乳球菌（Lactococcus Lactis)产生 的一种小分子肽，分子量约为 3510 D a。这种菌对许多革兰氏阳性菌， 尤其对引起食品腐败的芽孢杆菌、梭菌、葡萄球菌、利斯特氏菌等有 强烈抑制作用， 而乳酸链球菌素可被人所消化，故乳酸链球菌素作为 食品防腐剂具有天然无毒的特点，广泛用于乳制品、 罐装食品、 肉制 品和饮料的防腐保鲜剂。
杨博等人对乳链菌肽的最佳发酵条件进行了研究，其使用的菌株 为乳链菌肽生产菌株 (Lactococus lactis subsp. lactis ) Y - 72， 检测菌为黄色微球菌 （Micrococcus flavus) NCIB 8166由中科院微 生物研究所提供； 发酵培养基为： 大豆蛋白胨 1. 0%， 牛肉膏 0. 25%， 酵母膏 0. 25%, Na2HP04 · 12H021. 5%, MgS04 · 7H200. 012%,自然 PH值； 乳链菌肽效价检测培养基为：胰蛋白胨 0. 8%，酵母膏 0. 5%， NaClO. 5%, NaHP04 · 12 00. 2%,琼脂 1. 5%， PH6. 8; 所用试剂为： 乳链菌肽标准 品： Aplin & Barrett LTD生产， 胰蛋白胨： Oxoid LTD生产； 多胨： 日本产，其他培养基为北京市海淀区微生物培养基制品厂生产；其使 用方法为： 菌体生长测定： 用 721型分光光度计在 600nm处测定； 乳
链菌肽效价的测定：采用琼脂扩散法，测发酵液在检测平板上产生抑 菌圈的大小，用乳链菌肽标准品作对照，根据有关公式计算被测液的 效价。 作者从碳源种类、 碳源浓度、氮源、磷源、 磷源发酵量、 培养 温度、培养方式、接种量及 Y - 72发酵动力学方面对发酵条件进行了 研究（乳链菌肽的最佳发酵条件： 大连轻工业学院学报第 18卷第 1 期， 1999、 3 65-69)。
近年来， 人们探索用吸附剂吸附提取乳酸链球菌素的方法， Coventry等人用硅酸钙抗粘剂， 从乳酸乳球菌的发酵液中吸附乳酸 链球菌素， 但由于该法得到的是吸附复合物， 解吸困难。
Janes 等人用硅酸作吸附剂从乳酸乳球菌的发酵液中吸附乳酸 链球菌素时， 得到的是吸附复合液， 解吸仍困难。
00129928. X号中国专利申请披露了一种从乳酸乳球菌的发酵液 中吸附乳链菌肽的方法，其中记载的方法包括：用酸将乳酸乳球菌发 酵产生的含有乳链菌肽的发酵液调 PH至 1. 8-3. 5， 于 70- 100°C下加 热 1-60分钟， 杀死细菌并使乳链菌肽完全释放于发酵液中； 随后， 在 20-5CTC下加入为发酵液体积 0. 1-5%的吸附剂进行吸附，保温搅拌 0. 5- 3小时， 制成吸附剂-乳链菌肽复合物； 制成的吸附剂 -乳链菌肽 复合物的沉淀用蒸馏水洗涤两次， 然后用 10%- 20%发酵液体积的解吸 附剂进行解吸附，使用的解吸附剂为聚乙二醇与有机酸盐或无机酸盐 的水溶液， 在 20- 40°C下搅拌 5-60分钟进行解吸附。 该方法需吸附、 解吸附， 加入吸附剂和解吸附剂， 生产工艺复杂， 成本高，所需产物 活性易破坏，耗能高,活性保持低,效率低。
发明内容
本发明的目的是提供一种生产工艺简单、 成本低、 收率高，活性 保持高，分离效果好的乳酸链球菌素的分离提取方法。
为达到发明目的，本发明提供了一种从发酵液中分离乳酸链球菌 素的方法， 包括释放乳酸链球菌素、分离和喷雾干燥， 所说分离为采 用膜过滤的方法分别将分子量大于和小于乳酸链球菌素的分子量的 物质从发酵液中分离出去。
其中在温度 10-55Ό和压力 0. 5-2. 5 MPa条件下， 釆用无机膜或 管式膜过滤从发酵液中去除菌体和固体等分子量比乳酸链球菌素分 子量大的物质，其中无机膜或管式膜的孔径为 10- 60万，优选为 15-50 万； 温度优选为 15- 45°C， 最优选为 25- 40°C。
在温度 10- 55°C和压力 0. 5-2. 5 MPa条件下， 用卷式膜从发酵液 中去除分子量比乳酸链球菌素分子量小的物质， 其中卷式膜孔径为 0. 2 万， 优选为 1. 5- 3万； 温度优选为 15- 45°C , 最优选为 25 - 40 。C。
超滤后的发酵液浓缩物， 浓度为 100， 000- 500， 000 IU /ml, 加 入填料固体食盐， 使喷雾干燥后食盐含量为 10- 50%， 在喷雾千燥器 上进行喷雾干燥，热风进口温度为 120- 180°C，出口温度为 70- 100°C， 即得粉末状食品级的乳酸链球菌素产品。
注： 乳酸链球菌素即是乳链菌肽 nisin。 具体实施方式
另有说明除外， 本发明所使用的无机或管式膜系以金属氧化物、 陶瓷等为原料制作， 寿命较长， 膜主件主要由国外供应， 国内组装。 制造商有 Cerrer (法国）、 T0K (日本）、 Norton (美国）、 Mitsui (日 本）、 Nipongaishi (日本）， Sfec (法国）、 Corning (美国）、 DDS (丹 麦） 等公司。
卷式膜为有机材料制作， 主要有聚砜、 聚醚砜、 聚丙烯腈、 聚偏氟乙 烯、 醋酸纤维素等各种材料， 膜材主件一般由国外公司进口， 在国内 进行组装和配套，性能比较优越。制造商主要有 Pall (美国）、 Amicon (美国）、 HAYWARD (美国）等公司。
实施例 1
本实施例中，从乳酸乳球菌发酵液中提取乳酸链球菌素的具体步 骤如下：
1 ) 释放乳酸链球菌素：
首先取 200000 ml乳酸乳球菌发酵产生的含有乳酸链球菌素的 发酵液，用浓盐酸调 pH至 1. 8， 于 70°C下加热 60分钟， 使乳酸链球 菌素完全释放于发酵液中；
2) 膜过滤- 发酵液在温度为 15°C，操作压力为 0. 5MPa通过无机膜过滤而得 到去除菌体和固体等分子量比乳酸链球菌素分子量大的物质的发酵 液， 其中膜的孔径为 60万；
3) 卷式膜超滤：
步骤 2)所得到的发酵液在温度为 10°C， 操作压力为 0. 5MPa通
过卷式膜超滤， 去除分子量比乳酸链球菌素分子量小的物质，得到浓 度为 100， 000 IU/ml发酵液浓缩物， 其中卷式膜的孔径为 4万； 4) 喷雾干燥：
上述超滤后的发酵液浓缩物， 加入填料固体食盐， 使喷雾干燥 后食盐含量为 30%， 在喷雾干燥器上进行喷雾干燥， 热风进口温度为 120 °C , 出口温度为 70°C， 即得粉末状食品级的乳酸链球菌素产品。
其中无机膜组件为 Cerrer (法国）制造， 卷式膜组件为
HAYWARD (美国）制造。
经过测试， 整个流程乳酸链球菌素的收率为 89%。
实施例 2
本实施例中，从乳酸乳球菌发酵液中提取乳酸链球菌素的具体步 骤如下-
1 ) 释放乳酸链球菌素:
首先取 200000ml乳酸乳球菌发酵产生的含有乳酸链球菌素的发 酵液用浓硫酸调 pH至 3. 5， 于 100°C加热 1分钟，使乳酸链球菌素完 全释放于发酵液中；
2) 膜过滤：
发酵液在温度为 20°C，操作压力为 1. 5MPa通过无机膜过滤而得 到去除了菌体和固体等分子量比乳酸链球菌素分子量大的物质的发 酵液， 其中膜的孔径为 15万；
3) 卷式膜超滤：
步骤 2)所得到的发酵液在温度为 25°C， 操作压力为 2. 5MPa通
过卷式膜超滤， 去除分子量比乳酸链球菌素分子量小的物质，得到浓 度为 200, 000 IU/ml发酵液浓缩物， 其中卷式膜的孔径为 1. 5万； 4) 喷雾干燥：
上述超滤后的发酵液浓縮物， 加入填料固体食盐， 使喷雾干燥 后食盐含量为 40%， 在喷雾干燥器上进行喷雾干燥， 热风进口温度为 150°C， 出口温度为 80°C， 即得粉末状食品级的乳酸链球菌素产品。
其中无机膜组件为 T0K (日本）制造，卷式膜组件为 Ainicon (美 国）制造。
经过测试， 整个流程乳酸链球菌素的收率为 87%。
实施例 3
本实施例中，从乳酸乳球菌发酵液中提取乳酸链球菌素的具体步 骤如下-
1 )释放乳酸链球菌素：
首先取 200000ml乳酸乳球菌发酵产生的含有乳酸链球菌素的发 酵液用浓盐酸调 PH至 2. 0， 于 80°C加热 30分钟，使乳酸链球菌素完 全释放于发酵液中；
2)膜过滤- 发酵液在温度为 25°C，操作压力为 1. OMPa通过无机膜过滤而得 到去除了菌体和固体等分子量比乳酸链球菌素分子量大的物质的发 酵液， 其中膜的孔径为 20万；
3)卷式膜超滤- 步骤 2)所得到的发酵液在温度为 55°C， 操作压力为 1. 5MPa通
过卷式膜超滤， 去除分子量比乳酸链球菌素分子量小的物质，得到浓 度为 300, 000 IU/ml发酵液浓缩物， 其中卷式超滤膜的孔径为 1万； 4) 喷雾干燥：
上述超滤后的发酵液浓缩物， 加入填料固体食盐， 使喷雾干燥 后食盐含量为 50%， 在喷雾干燥器上进行喷雾干燥， 热风进口温度为 180°C， 出口温度为 100°C， 即得粉末状食品级的乳酸链球菌素产品。
其中无机膜组件为 Norton (美国） 制造， 卷式膜组件为
HAYWARD (美国）制造。
经过测试， 整个流程乳酸链球菌素的收率为 85%。
实施例 4
本实施例中，从乳酸乳球菌发酵液中提取乳酸链球菌素的具体步 骤如下：
1 )释放乳酸链球菌素：
首先取 200000ml乳酸乳球菌发酵产生的含有乳酸链球菌素的发 酵液用浓盐酸调 PH至 2. 5， 于 85°C加热 20分钟，使乳酸链球菌素完 全释放于发酵液中；
2)膜过滤：
发酵液在温度为 30°C，操作压力为 1. 2MPa通过管式膜过滤而得 到去除了菌体和固体等分子量比乳酸链球菌素分子量大的物质的发 酵液， 其中膜的孔径为 10万；
3)卷式膜超滤：
步骤 2)所得到的发酵液在温度为 20°C， 操作压力为 1. OMPa通
过卷式膜超滤， 去除分子量比乳酸链球菌素分子量小的物质，得到浓 度为 500， 000 IU/ml发酵液浓缩物， 其中卷式膜的孔径为 2万； 4) 喷雾干燥：
上述超滤后的发酵液浓缩物， 加入填料固体食盐， 使喷雾干燥 后食盐含量为 10%， 在喷雾干燥器上进行喷雾干燥， 热风进口温度为 160°C， 出口温度为 90°C， 即得粉末状食品级的乳酸链球菌素产品。
其中无机膜组件为 Mitsui (日本）制造，卷式膜组件为 Pall (美 国）制造。
经过测试， 整个流程乳酸链球菌素的收率为 87%。
实施例 5
本实施例中，从乳酸乳球菌发酵液中提取乳酸链球菌素的具体步 骤如下：
1 )释放乳酸链球菌素- 首先取 200000ml乳酸乳球菌发酵产生的含有乳酸链球菌素的发 酵液用浓硫酸调 pH至 3. 0，于 90°C加热 45分钟，使乳酸链球菌素完 全释放于发酵液中；
2)膜过滤：
发酵液在温度为 55°C，操作压力为 0. 5MPa通过管式膜过滤而得 到去除了菌体和固体等分子量比乳酸链球菌素分子量大的物质的发 酵液， 其中膜的孔径为 30万；
3)卷式膜超滤：
步骤 2)所得到的发酵液在温度为 18°C， 操作压力为 2. 0MPa通
过卷式膜超滤， 去除分子量比乳酸链球菌素分子量小的物质，得到浓 度为 400, 000 IU/ml发酵液浓缩物， 其中卷式膜的孔径为 3万； 4) 喷雾干燥：
上述超滤后的发酵液浓缩物， 加入填料固体食盐， 使喷雾干燥 后食盐含量为 50%。 在喷雾干燥器上迸行喷雾干燥， 热风进口温度为 165° (：， 出口温度为 85°C， 即得粉末状食品级的乳酸链球菌素产品。
其中无机膜组件为 Nipongaishi (日本)制造， 卷式膜组件为 Ami con (美国）制造。
经过测试， 整个流程乳酸链球菌素的收率为 85%。
实施例 6
本实施例中，从乳酸乳球菌发酵液中提取乳酸链球菌素的具体步 骤如下：
1 )释放乳酸链球菌素- 首先取 200000ml乳酸乳球菌发酵产生的含有乳酸链球菌素的发 酵液用浓盐酸调 pH至 3. 5， 于 100°C加热 1分钟，使乳酸链球菌素完 全释放于发酵液中；
2)膜过滤：
发酵液在温度为 10°C，操作压力为 1. 5MPa通过无机膜过滤而得 到去除了菌体和固体等分子量比乳酸链球菌素分子量大的物质的发 酵液， 其中膜的孔径为 45万；
3)卷式膜超滤：
步骤 2)所得到的发酵液在温度为 30°C， 操作压力为 1. 2MPa通
过卷式膜超滤， 去除分子量比乳酸链球菌素分子量小的物质，得到浓 度为 250， 000 IU/ml发酵液浓缩物， 其中卷式膜的孔径为 2. 5万； 4) 喷雾干燥：
上述超滤后的发酵液浓缩物， 在加入填料固体食盐， 使喷雾干 燥后食盐含量为 20%, 在喷雾干燥器上进行喷雾干燥， 热风进口温度 为 150°C，出口温度为 80°C，即得粉末状食品级的乳酸链球菌素产品。
其中无机膜组件为 DDS (丹麦）制造，卷式膜组件为 HAYWARD (美 国）制造。
经过测试， 整个流程乳酸链球菌素的收率为 86%。
实施例 7
本实施例中，从乳酸乳球菌发酵液中提取乳酸链球菌素的具体步 骤如下-
1 ) 释放乳酸链球菌素：
首先取 200000ml乳酸乳球菌发酵产生的含有乳酸链球菌素的发 酵液用浓硫酸调 pH至 3. 5， 于 100°C加热 1分钟，使乳酸链球菌素完 全释放于发酵液中；
2) 膜过滤：
发酵液在温度为 10°C，操作压力为 1. 6MPa通过无机膜过滤而得 到去除了菌体和固体等分子量比乳酸链球菌素分子量大的物质的发 酵液， 其中膜的孔径为 50万；
3)卷式膜超滤- 步骤 2)所得到的发酵液在温度为 40°C， 操作压力为 2. 5MPa通
过卷式膜超滤， 去除分子量比乳酸链球菌素分子量小的物质，得到浓 度为 350， 000 IU/ml发酵液浓缩物， 其中卷式膜的孔径为 1. 5万； 4) 喷雾干燥：
上述超滤后的发酵液浓缩物， 在加入填料固体食盐， 使喷雾干 燥后食盐含量为 45%， 在喷雾干燥器上进行喷雾干燥， 热风进口温度 为 150°C，出口温度为 80°C，即得粉末状食品级的乳酸链球菌素产品。
其中无机膜组件为 Corning (美国）制造， 卷式膜组件为 Amicon (美国）制造。
经过测试， 整个流程乳酸链球菌素的收率为 85%。
本发明提供的从发酵液中分离提取乳酸链球菌素的方法， 由于 采用无机膜过滤和卷式膜超滤， 生产工艺简单、成本大大降低， 且对 目标产物无破坏,活性保持高。
其中收率计算方法：
总收率％=成品效价 X成品重量 I发酵液效价 X发酵液体积 X
%。
Claims
权利 要求 、 一种从发酵液中分离乳酸链球菌素的方法， 所述方法包括释放乳 酸链球菌素、 分离和喷雾干燥， 其特征在于： 所说分离为采用膜 过滤的方法分别将分子量大于和小于乳酸链球菌素的分子量的物 质从发酵液中分离出去。
、权利要求 1的分离乳酸链球菌素的方法，其特征在于在温度 10 - 55 °C和压力 0. 5-2. 5 MPa条件下， 采用无机膜或管式膜过滤从发酵液 中去除菌体和固体等分子量比乳酸链球菌素分子量大的物质；用卷 式膜超滤从发酵液中去除分子量比乳酸链球菌素分子量小的物质， 得到浓度为 100， 000- 500, 000 IU/ml发酵液浓缩物。
、 根据权利要求 2所述的分离乳酸链球菌素的方法， 其特征在于无 机膜或管式膜的孔径为 10- 60万。
、 根据权利要求 3所述的分离乳酸链球菌素的方法， 其特征在于无 机膜或管式膜的孔径为 15-50万。
、 根据权利要求 2所述的分离乳酸链球菌素的方法， 其特征在于温 度为 15- 45°C。
、 根据权利要求 5所述的分离乳酸链球菌素的方法， 其特征在于温 度为 25- 40°C。
、 根据权利要求 2所述的分离乳酸链球菌素的方法， 其特征在于卷 式膜的孔径为 0. 2- 4万。
、 根据权利要求 7所述的分离乳酸链球菌素的方法， 其特征在于卷
式膜的孔径为 1. 5- 3万。
、 根据权利要求 1所述的分离乳酸链球菌素的方法， 其特征在于超 滤后的发酵液浓缩物，加入填料固体食盐，使喷雾干燥后食盐含量 为 10-50%， 在喷雾干燥器上进行喷雾干燥， 热风进口温度为 120-180 °C， 出口温度为 70- 100°C， 即得粉末状食品级的乳酸链 球菌素产品。
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2005100664817A CN1293092C (zh) | 2005-04-26 | 2005-04-26 | 从乳酸乳球菌发酵液中分离乳酸链球菌素的方法 |
| CN200510066481.7 | 2005-04-26 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO2006114039A1 true WO2006114039A1 (fr) | 2006-11-02 |
Family
ID=35305152
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/CN2006/000638 WO2006114039A1 (fr) | 2005-04-26 | 2006-04-11 | Procede de separation de nisine d'un liquide de fermentation de galactococcus lactique |
Country Status (2)
| Country | Link |
|---|---|
| CN (1) | CN1293092C (zh) |
| WO (1) | WO2006114039A1 (zh) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116046916A (zh) * | 2021-02-03 | 2023-05-02 | 仲景宛西制药股份有限公司 | 一种黄芪桂枝五物汤制剂的质量控制方法 |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101113463B (zh) * | 2007-05-23 | 2010-05-26 | 齐齐哈尔安泰生物工程有限公司 | 一种乳酸链球菌素的制备方法 |
| CN102286393B (zh) * | 2011-03-01 | 2014-04-30 | 安徽农业大学 | 一种乳酸乳球菌和该乳酸乳球菌产生的抗菌肽以及该抗菌肽的用途 |
| CN105713929B (zh) * | 2016-03-29 | 2019-02-19 | 安泰生物工程股份有限公司 | 一种利用Nisin发酵废菌体抑制乙醇发酵过程中乳酸菌生长的方法 |
| CN111662365A (zh) * | 2020-05-28 | 2020-09-15 | 河北圣雪大成制药有限责任公司 | 一种乳酸链球菌素的提取方法 |
| CN113201053B (zh) * | 2021-05-10 | 2023-03-07 | 南京钛净流体技术有限公司 | 一种膜分离提取乳酸菌细菌素的方法及装置 |
| CN115386523B (zh) * | 2022-09-30 | 2024-01-30 | 北京诺安百汇医药科技有限公司 | 一株乳酸乳球菌及其在抗幽门螺杆菌感染的应用 |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0475596A (ja) * | 1990-07-16 | 1992-03-10 | Daicel Chem Ind Ltd | ナイシンの製造方法 |
| CN1293201A (zh) * | 2000-10-18 | 2001-05-02 | 中国科学院微生物研究所 | 从发酵液中分离提取乳链菌肽的方法 |
-
2005
- 2005-04-26 CN CNB2005100664817A patent/CN1293092C/zh active Active
-
2006
- 2006-04-11 WO PCT/CN2006/000638 patent/WO2006114039A1/zh active Application Filing
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0475596A (ja) * | 1990-07-16 | 1992-03-10 | Daicel Chem Ind Ltd | ナイシンの製造方法 |
| CN1293201A (zh) * | 2000-10-18 | 2001-05-02 | 中国科学院微生物研究所 | 从发酵液中分离提取乳链菌肽的方法 |
Non-Patent Citations (2)
| Title |
|---|
| HIROSHI S. ET AL.: "Nisin Production by a Mixed-Culture System Consisting of Lactococcus lactis and Kluyveromyces marxianus", APPL. ENVIRON. MICROBIOL., vol. 65, no. 7, July 1999 (1999-07-01), pages 3134 - 3141, XP003002120 * |
| ZHANG Y. ET AL.: "Membrane Separation Technology and its Application in the Downstream Process of Fermentation Liquors", JOURNAL OF HEBEI UNIVERSITY OF SCIENCE AND TECHNOLOGY, vol. 23, no. 3, 2002, pages 21 - 26 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116046916A (zh) * | 2021-02-03 | 2023-05-02 | 仲景宛西制药股份有限公司 | 一种黄芪桂枝五物汤制剂的质量控制方法 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1687110A (zh) | 2005-10-26 |
| CN1293092C (zh) | 2007-01-03 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| WO2006114039A1 (fr) | Procede de separation de nisine d'un liquide de fermentation de galactococcus lactique | |
| Martinez et al. | Bacteriocin production by Bifidobacterium spp. A review | |
| Ogunbanwo et al. | Characterization of bacteriocin produced by Lactobacillus plantarum F1 and Lactobacillus brevis OG1 | |
| CN104531562B (zh) | 一种植物乳杆菌植物亚种及其抗单增李斯特菌细菌素的制备方法 | |
| CN101294188B (zh) | 乳铁蛋白抗菌肽及其制备方法与它们的用途 | |
| Jamuna et al. | Isolation and characterization of lactobacilli from some traditional fermented foods and evaluation of the bacteriocins | |
| Bortner et al. | Bactericidal effect of lactoferrin on Legionella pneumophila | |
| CN102286393B (zh) | 一种乳酸乳球菌和该乳酸乳球菌产生的抗菌肽以及该抗菌肽的用途 | |
| Ahmed et al. | Lactobacillus acidophilus bacteriocin, from production to their application: An overview | |
| Naimah et al. | Isolation, purification and characterization of antimicrobial peptides produced from Saccharomyces boulardii | |
| Yıldırım et al. | Factors affecting the adsorption of buchnericin LB, a bacteriocin produced by Lactocobacillus buchneri | |
| Zhijing et al. | Screening beneficial bacteriostatic lactic acid bacteria in the intestine and studies of bacteriostatic substances | |
| Singh et al. | Antimicrobial activity of bioactive peptides derived from fermentation of soy milk by Lactobacillus plantarum C2 against common foodborne pathogens | |
| CN103724400A (zh) | 一种分离纯化脱水达托霉素的方法 | |
| Yang et al. | Antimicrobial activity of 2-pyrrolidone-5-carboxylic acid produced by lactic acid bacteria | |
| KR20190009458A (ko) | 바실러스 서브틸리스를 이용한 오크라톡신 a 저감 방법 | |
| CN103059105B (zh) | 硫酸粘菌素的提取方法 | |
| CN102304172B (zh) | 一种乳酸链球菌素的提取方法 | |
| CN104987370A (zh) | 一种细菌素及其制备方法和应用 | |
| CN110746488A (zh) | 一种具有食品防腐保鲜作用的细菌素pe-zyb1及其应用 | |
| CN107056903B (zh) | 一种利用琼脂糖凝胶亲和层析色谱提取医药级超高纯度乳酸链球菌素的方法 | |
| CN111499703A (zh) | 一种高收率乳酸链球菌素的提取方法 | |
| CN105755074A (zh) | 一种Nisin的提取方法 | |
| CN102550799B (zh) | 一种从发酵液中快速提取食用抗菌肽的方法 | |
| DÜNDAR et al. | Large-scale purification of a bacteriocin produced by Leuconostoc mesenteroides subsp. cremoris using diatomite calcium silicate |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| 121 | Ep: the epo has been informed by wipo that ep was designated in this application | ||
| NENP | Non-entry into the national phase |
Ref country code: DE |
|
| WWW | Wipo information: withdrawn in national office |
Country of ref document: DE |
|
| NENP | Non-entry into the national phase |
Ref country code: RU |
|
| WWW | Wipo information: withdrawn in national office |
Country of ref document: RU |
|
| 122 | Ep: pct application non-entry in european phase |
Ref document number: 06722290 Country of ref document: EP Kind code of ref document: A1 |