WO2017016199A1 - 一种沙链霉菌的用途及香兰素的生产方法 - Google Patents

一种沙链霉菌的用途及香兰素的生产方法 Download PDF

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WO2017016199A1
WO2017016199A1 PCT/CN2016/070788 CN2016070788W WO2017016199A1 WO 2017016199 A1 WO2017016199 A1 WO 2017016199A1 CN 2016070788 W CN2016070788 W CN 2016070788W WO 2017016199 A1 WO2017016199 A1 WO 2017016199A1
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fermentation
vanillin
medium
seed
cultured
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赵希景
邢晨光
黄志强
刘伟
龚宏灿
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厦门欧米克生物科技有限公司
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Priority to EP16829569.9A priority Critical patent/EP3339443B1/en
Priority to US15/575,264 priority patent/US10364446B2/en
Publication of WO2017016199A1 publication Critical patent/WO2017016199A1/zh

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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
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    • C12R2001/465—Streptomyces

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  • the present invention relates to the use of S. sphaeroides and a method for producing vanillin.
  • Vanillin also known as vanillin, has a molecular weight of 152.14. White to pale yellow crystal, melting point 81 ° C, boiling point 284-285 ° C, relative density 1.060, flash point 147 ° C, solubility: 10g / L (25 ° C), soluble in organic solvents such as ethanol. Found in Peru balsam, scented geranium oil, vanilla, coffee, grapes, brandy, with a vanilla flavor, very sweet taste.
  • Vanillin is not only an important spice, but also an important chemical raw material, and an important pharmaceutical intermediate. In foods, because of its aroma of vanilla beans, it is used in a variety of foods as a fixative. In medicine, vanillin is a raw material for the synthesis of pharmaceutical intermediates, which can be used to produce raw materials for the treatment of high blood pressure, heart disease and skin diseases.
  • Lignin method Lignin is widely present in waste wood, pulp waste liquid, and can be obtained by hydrolysis, oxidation and other reactions in an alkaline medium. However, although the raw material cost of the method is low, the production of vanillin is low, and the product quality is low, and generally cannot be used in the food and pharmaceutical industries.
  • Guaiacol method There are several ways to synthesize vanillin by guaiacol method, the highest yield is 60%, generally less than 60%. This method is the main production method of domestic vanillin.
  • Baicalein is mainly derived from camphor oil. This reaction route is long and has by-products and is not easy to separate.
  • the plant extraction method mainly uses vanilla bean to extract, but since the cultivation of vanilla bean is limited by geographical conditions, and the content of vanillin in vanilla bean is not high, the fragrance extracted from vanilla bean is Because of its low output, Lansu can't meet the market demand, and the cost is high, which is difficult for the average consumer to accept.
  • Biotransformation methods mainly include plant cell culture methods and microbial transformation methods.
  • the plant cell culture method has a relatively long incubation period and a low yield, which is not conducive to industrialization.
  • the substrate is mainly ferulic acid or isoeugenol. Microbial transformation method, short cycle, high yield, less pollution is more conducive to industrial production.
  • lipoxygenase can catalyze the conversion of eugenol or isoeugenol to vanillin.
  • the reaction conditions are mild and the product is easy to purify.
  • the enzyme separation and the directional transformation of the enzyme, the modification needs further research, and the method is still in the research stage.
  • a method for producing vanillin which is produced by using Streptomyces psammoticus OMK-4.
  • a method for producing vanillin characterized in that the production method is fermentation.
  • a method for producing vanillin comprises the following steps:
  • the solid slant medium formulation consisted of the following ingredients: Solid slant medium formulation as follows: (%)
  • Seed culture Under sterile conditions, a well-grown strain is inserted into the sterile seed culture medium from the cultured solid slope by using the inoculating shovel. The initial pH of the seed medium is 5-8. The cells are cultured to a logarithmic growth phase at a temperature of 28-35 ° C and a rotational speed of 200-500 rpm;
  • the seed medium formulation includes the following components (%): soluble starch 1.0-3.5, potassium dihydrogen phosphate 0.1-0.5, urea 0.1-0.3, magnesium sulfate 0.05-0.1, calcium carbonate 0.1-0.3, yeast dip 0.1-1.0, corn Slurry 0.1-1.0, ammonium sulfate 0.1-0.6, ferulic acid 0.1-0.3;
  • Fermentation the seed liquid cultured to the logarithmic growth phase is introduced into the fermentation medium under a sterile condition at a volume ratio of 5-15%; the initial pH of the fermentation medium is 7.2-7.8 at a temperature of 30-40 °C, stirring speed 200-500rpm, aeration of 1:0.5 under the conditions of fermentation 70-120h; fermentation formula includes the following components (%):
  • Soluble starch 2.0-5.0, potassium dihydrogen phosphate 0.1-0.3, urea 0.1-0.5, magnesium sulfate 0.05-0.1, calcium carbonate 0.5-2.0, yeast dip 0.1-1.0, ammonium sulfate 0.1-0.5, ferulic acid 1-3 .
  • the desired vanillin is extracted from the fermentation broth.
  • the desired vanillin is extracted from the fermentation broth.
  • the fermentation broth was heated to 80 ° C, and a ceramic membrane was filtered to remove some impurities such as bacteria and macromolecules.
  • the filtrate is subjected to ultrafiltration (UF) to remove small molecular proteins, pigments and other impurities, and finally the ultrafiltrate is subjected to reverse osmosis (RO) for concentration, concentrated 5-10 times, the concentration of the concentrated solution is adjusted to 5-6, and the crystal is cooled and crystallized.
  • UF ultrafiltration
  • RO reverse osmosis
  • the Streptomyces psammoticus OMK-4 has been deposited with the China Center for Type Culture Collection on May 27, 2015. The deposit is located in Wuhan, Hubei province, Wuhan University, and the deposit number is CCTCC M 2015329. .
  • the applicant first discovered that the S. cerevisiae strain has the ability to produce vanillin and applies it to the production of vanillin.
  • the invention adopts a fermentation method to produce vanillin, and adopts a raw material ferulic acid and the like to generate a target product through microbial metabolism.
  • the process belongs to low temperature and low pressure, is safe, simple in operation and less in pollution, and is a safe and environmentally friendly production method. .
  • Figure 1 is an HPLC chart demonstrating that the resulting product is vanillin.
  • a strain of Streptomyces psammoticus OMK-4 with lauric acid production capacity was isolated from the soil under the cinnamon plantation forest.
  • the strain was deposited with the China Center for Type Culture Collection on May 27, 2015, and the deposit number is CCTCC M 2015329. .
  • the gram-positive bacteria of this bacterium are poorly grown on the medium of C. chinensis.
  • the nematode chain is observed under the light microscope, and the spores are bone-shaped.
  • the glycogen and dextrin can be used, and D-fructose and D-fiber can not be used.
  • Carbon source such as disaccharide.
  • the 16S rRNA gene sequencing result was 100% coincident with S. serrata and identified as S.
  • the solid slant medium formulation is as follows: (%)
  • a well-grown strain from a cultured solid slope is placed in a sterile seed culture medium with an initial pH of 5-8 at a culture temperature of 28-35 ° C.
  • the cells were cultured to a logarithmic growth phase at a rotational speed of 150-500 rpm.
  • the seed medium formula is as follows: (%)
  • the seed liquid cultured to the logarithmic growth phase was introduced into the fermentation medium under a sterile condition at a volume ratio of 10%.
  • the initial pH of the fermentation medium was 7.5, and the fermentation was carried out for 70-120 h at a temperature of 30-40 ° C, a stirring speed of 200-500 rpm, and an aeration rate of 1:0.5.
  • the fermentation formula is as follows: (%)
  • Soluble starch 2.0-5.0, potassium dihydrogen phosphate 0.1-0.3, urea 0.1-0.5, magnesium sulfate 0.05-0.1, calcium carbonate 0.5-2.0, yeast dip 0.1-1.0, ammonium sulfate 0.1-0.5, ferulic acid 2.0.
  • the seed medium was prepared, and the medium composition was as follows (g/L): glucose 20, potassium dihydrogen phosphate 3, urea 5, magnesium sulfate 0.3, sodium chloride 1.0, yeast dip 5, corn syrup 5, ammonium sulfate 6
  • the calcium carbonate 0.2 solvent is water, and the initial pH is 7.2. Sterilization at 121 ° C for 20 min.
  • the fermentation medium is prepared, and the medium composition is as follows (g/L): glucose 30, potassium dihydrogen phosphate 5, urea 5, magnesium sulfate 0.6, yeast dip powder 8, ammonium sulfate 3, calcium carbonate 1.0 ferulic acid 20, solvent For water, the initial pH was 7.2. Sterilize at 121 ° C for 20 min.
  • Fermentation of natural vanillin 50 mL of the prepared fermentation medium is poured into a 500 mL sterile flask, and then 7.5 mL of the cultured seed solution is added for fermentation, the fermentation temperature is 37 ° C, and the shaking speed is 200 rpm. After 80 hours of fermentation, the concentration of natural vanillin in the fermentation broth was determined by HPLC to be 10 g/L.
  • the seed medium and the fermentation medium were prepared in the same manner as in the example 1.
  • Fermentation of natural vanillin 10.2L of the prepared fermentation medium is charged into a 20L fermenter, 121 After sterilization at °C for 20 min, the cultured 1.8 L seed solution was then introduced into a 20 L fermentor for fermentation at a fermentation temperature of 37 ° C, a stirring speed of 400 rpm, and aeration ratio of 1:0.2. The fermentation cycle is 90h. At the end of the fermentation, the concentration of natural vanillin in the fermentation broth was determined by HPLC to be 15 g/L. (The HPLC chart is shown in Figure 1)
  • the fermentation broth of the natural vanillin concentration of 15 g / L of Example 2 was 12 L, and the membrane was filtered, 10 L of the filtrate was subjected to ultrafiltration (UF), and the ultrafiltrate was subjected to reverse osmosis (RO) to finally obtain 5L concentrated solution, the concentration of the concentrated solution was adjusted to 5-6, and the temperature was lowered to 0-4 ° C for 4 hours.
  • the crude natural vanillin was obtained by suction filtration, and dried to obtain 144 g of crude product with a purity of 98%.
  • the invention adopts the fermentation method to produce vanillin, which belongs to low temperature and low pressure, is relatively safe, has simple operation and less pollution.

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Abstract

本申请提供了一种沙链霉菌（Streptomyces psammoticus OMK-4）及用该菌株生产香兰素的方法，所述方法包括如下步骤：菌种活化、种子培养、发酵、提取等，原料采用阿魏酸，通过微生物发酵法生产香兰素，安全环保。

Description

一种沙链霉菌的用途及香兰素的生产方法 技术领域
本发明涉及一种沙链霉菌的用途及香兰素的生产方法。
背景技术
香兰素(Vanillin)，又名香草醛，分子量：152.14。白色至浅黄色晶体，熔点81℃，沸点284-285℃，相对密度1.060，闪点147℃，溶解性：10g/L(25℃)，溶于乙醇等有机溶剂。在秘鲁香脂，丁子香芽油，香子兰，咖啡，葡萄，白兰地中有发现，具有香子兰气味，十分甜的味道。
香兰素不仅是一种重要的香料，还是一种重要的化工原料，又是一种重要的医药中间体。在食品上，因其具有香荚兰豆的香气，大量用于各种食品中用做定香剂。在医药上，香兰素是合成医药中间体的原料，可用来生产治疗高血压、心脏病，皮肤病常用药物的原料。
1、化学合成法
1)木质素法：木质素广泛存在于废木材，纸浆废液中，在碱性介质中经水解，氧化等反应可得到香兰素。但该法原料成本虽低，香兰素产量也低，且产品质量偏低，一般不能用于食品和药品工业。
2)丁香酚法：丁香酚为丁香油的主要成分，经氧化等反应后可得到香兰素。
3)愈创木酚法：愈创木酚法合成香兰素有多条途径，最高收率达到60％，一般小于60％。该方法是国内香兰素的主要生产方法。
4)对羟基苯甲醛、对硝基甲苯法、对甲酚法：这三种方法由于种种原因还没有工业化生产(对羟基苯甲醛由于原料价格高；对甲酚法反应条件苛刻，收率低；对硝基甲苯法需要设备比较大)。
5)黄樟素法：黄樟素主要来源于樟脑油，此反应路线较长，且有副产物，不易分离。
2、植物提取法
植物提取法主要是用香荚兰豆进行提取，但由于香荚兰豆的种植受地理条件限制，而且香荚兰豆中香兰素的含量不高，因此从香荚兰豆中提取的香兰素由于产量低，满足不了市场的需求，成本高，一般消费者难以接受。
3、生物转化法
生物转化法主要有植物细胞培养法和微生物转化法。植物细胞培养法培养周期相对较长，且产率不高，不利于工业化。微生物转化法所用的微生物有多种，底物主要是阿魏酸或异丁香酚。微生物转化法，周期短，产率高，污染少比较有利于工业化生产。
4、酶法
目前发现脂氧合酶可以催化丁香酚或异丁香酚转化为香兰素，该反应条件温和，产物易纯化。但是酶分离和酶的定向改造，修饰还需要进一步研究，该法还处于研究阶段。
综上，以上方法分别存在各种不足之处。
发明内容
本发明的目的在于提供一种沙链霉菌的用途及香兰素的生产方法，以解决现有技术中存在的上述问题。
本发明提供的技术方案如下：
一种沙链霉菌Streptomyces psammoticus OMK-4的用途，其用于生产香兰素。
一种香兰素的生产方法，其特征在于：其是利用沙链霉菌Streptomyces psammoticus OMK-4生产。
一种香兰素的生产方法，其特征在于：所述的生产方法为发酵。
一种香兰素的生产方法，包括如下步骤：
1)在无菌条件下从甘油管中划一满环Streptomyces psammoticus OMK-4均匀涂布于固体斜面培养基上，在生化培养箱中于26-30℃培养24-48h；其中， 所述的菌液为沙链霉菌；
固体斜面培养基配方包括如下成分：固体斜面培养基配方如下：(％)
可溶性淀粉1.0-3.5，磷酸二氢钾0.1-1.0，氯化钠0.05-0.3，酵母浸粉0.1-1.0；
2)种子培养：在无菌条件下从培养好的固体斜面上用接种铲划一满环长势良好的菌株接入无菌种子培养基中，该种子培养基初始pH值为5-8，在培养温度28-35℃，转速200-500rpm的条件下培养菌体到对数生长期；
种子培养基配方包括如下成分(％)：可溶性淀粉1.0-3.5、磷酸二氢钾0.1-0.5、尿素0.1-0.3、硫酸镁0.05-0.1、碳酸钙0.1-0.3、酵母浸粉0.1-1.0、玉米浆0.1-1.0、硫酸铵0.1-0.6、阿魏酸0.1-0.3；
3)、发酵：将培养至对数生长期的种子液在无菌条件下以5-15％的体积比接入发酵培养基；发酵培养基初始pH值为7.2-7.8，在温度30-40℃，搅拌转速200-500rpm，通气量1:0.5的条件下发酵70-120h；发酵配方包括如下组分(％)：
可溶性淀粉2.0-5.0、磷酸二氢钾0.1-0.3、尿素0.1-0.5、硫酸镁0.05-0.1、碳酸钙0.5-2.0、酵母浸粉0.1-1.0、硫酸铵0.1-0.5、阿魏酸1-3。
4)提取：从发酵液中提取所需的香兰素。从发酵液中提取所需的香兰素。发酵结束，将发酵液升温至80℃，进行陶瓷膜过滤，去除菌体、大分子蛋白等一些杂质。然后滤液进行超滤(UF)去除小分子蛋白，色素等杂质，最后超滤液进行反渗透(RO)进行浓缩，浓缩5-10倍，将浓缩液PH调至5-6，降温结晶，得香兰素粗品。
在本发明中，该沙链霉菌(Streptomyces psammoticus OMK-4)已于2015年5月27日保藏于中国典型培养物保藏中心，保藏地点为湖北省武汉市，武汉大学，保藏号为CCTCC M 2015329。
申请人首次发现该沙链霉菌菌株具有生产香兰素的能力，并将其应用于香兰素的生产。
本发明采用发酵法生产香兰素，采用原料阿魏酸等，经过微生物代谢生成目标产物的一种方法，该工艺属于低温低压，比较安全，操作简单，污染少，是一个安全环保的生产方法。
附图说明
图1为HPLC图，证明所得的产物为香兰素。
具体实施方式
1、菌种
从肉桂种植园树林下土壤中分离出一株具有桂酸生产能力的菌株Streptomyces psammoticus OMK-4，该菌已于2015年5月27日保藏于中国典型培养物保藏中心，保藏号为CCTCC M 2015329。该菌革兰氏阳性菌，在察氏洋菜培养基上生长较差，光学显微镜下观察到直孢子链，孢子指骨状；能利用糖原、糊精，不能利用D-果糖，D-纤维二糖等碳源。16S rRNA基因测序结果与沙链霉菌100％吻合，鉴定为沙链霉菌。
1、菌种活化
在无菌条件下从甘油管中划一满环菌液均匀涂布固体斜面培养基上，在生化培养箱中于28-30℃培养24-48h。固体斜面培养基配方如下：(％)
可溶性淀粉1.0-3.5，磷酸二氢钾0.1-1.0，氯化钠0.05-0.3，酵母浸粉0.1-1.0
2、种子培养
在无菌条件下从培养好的固体斜面上用接种铲划一满环长势良好的菌株接入无菌种子培养基中，该种子培养基初始PH值为5-8，在培养温度28-35℃，转速150-500rpm的条件下培养菌体到对数生长期。种子培养基配方如下：(％)
可溶性淀粉1.0-3.5、磷酸二氢钾0.1-0.5、尿素0.1-0.3、硫酸镁0.05-0.1、碳酸钙0.1-0.3、酵母浸粉0.1-1.0、玉米浆0.1-1.0、硫酸铵0.1-0.6和阿魏酸0.2。
3、发酵
将培养至对数生长期的种子液在无菌条件下以10％的体积比接入发酵培养基。发酵培养基初始PH值为7.5，在温度30-40℃，搅拌转速200-500rpm，通气量1:0.5的条件下发酵70-120h。发酵配方如下：(％)
可溶性淀粉2.0-5.0、磷酸二氢钾0.1-0.3、尿素0.1-0.5、硫酸镁0.05-0.1、碳酸钙0.5-2.0、酵母浸粉0.1-1.0、硫酸铵0.1-0.5、阿魏酸2.0。
实施例1：天然香兰素的生产(三角瓶震荡发酵)
配制种子培养基，其培养基组成如下(g/L)：葡萄糖20，磷酸二氢钾3，尿素5，硫酸镁0.3，氯化钠1.0，酵母浸粉5，玉米浆粉5，硫酸铵6，碳酸钙0.2溶剂为水，初始PH值为7.2。121℃灭菌20min。
配制发酵培养基，其培养基组成如下(g/L)：葡萄糖30，磷酸二氢钾5，尿素5，硫酸镁0.6，酵母浸粉8，硫酸铵3，碳酸钙1.0阿魏酸20，溶剂为水，初始PH值为7.2。121℃灭菌20min。
种子的制备：无菌转移低温甘油管OMK-4菌株于新鲜、无菌固体平板上，30℃活化2天，挑取菌落接种于500mL三角瓶做种子培养，瓶中种子培养基的体积为50mL，摇床转速180rpm，30℃培养16～24h，得到种子液。
天然香兰素的发酵：取配制好的发酵培养基50mL倒入500mL的无菌三角瓶中，然后接入培养好的种子液7.5mL进行发酵，发酵温度37℃，摇床转速200rpm。发酵80h后用HPLC法测定发酵液中天然香兰素的浓度为10g/L。
实施例2：天然香兰素的生产(搅拌反应器发酵)
种子培养基、发酵培养基配制同实施例1.
种子的制备：无菌转移低温甘油管OMK-4菌株于新鲜、无菌固体平板上，30℃活化2天，挑取菌落接种于装有1.8L种子培养基的3L种子罐进行种子培养，通风搅拌转速300rpm，30℃培养13-18h，得到种子液。
天然香兰素的发酵：将配制好的发酵培养基10.2L装入20L发酵罐中，121 ℃灭菌20min，然后将培养好的1.8L种子液接入20L发酵罐中进行发酵，发酵温度37℃，搅拌转速400rpm，通气比1:0.2。发酵周期90h。发酵结束用HPLC法测定发酵液中天然香兰素的浓度为15g/L。(其中HPLC图见图1)
实施例3:天然香兰素的提取
取温度为80℃，实施例2天然香兰素浓度为15g/L的发酵液12L，进行陶瓷膜过滤，将10L滤液进行超滤(UF)，超滤液进行反渗透(RO)，最终得到5L浓缩液，将浓缩液调PH值至5-6，降温至0-4℃结晶4h，抽滤得天然香兰素粗品，烘干得144g粗品，纯度为98％。
工业实用性
本发明采用发酵法生产香兰素，属于低温低压，比较安全，操作简单，污染少。

Claims (8)

  1. 一种沙链霉菌Streptomyces psammoticus OMK‐4的用途，其用于生产香兰素。
  2. 一种香兰素的生产方法，其特征在于：其是利用沙链霉菌Streptomyces psammoticus OMK‐4生产。
  3. 如权利要求2所述的一种香兰素的生产方法，其特征在于：所述的生产方法为发酵。
  4. 如权利要求2或3的一种香兰素生产方法，其特征在于采用原料阿魏酸，经过微生物代谢生成目标产物的。
  5. 一种香兰素的生产方法，包括如下步骤：
    1)在无菌条件下从甘油管中划一满环菌液均匀涂布于固体斜面培养基上，在生化培养箱中于26-30℃培养24-48h；其中，所述的菌液为沙链霉菌Streptomyces psammoticus OMK‐4；
    固体斜面培养基配方按重量百分比包括如下成分：
    可溶性淀粉1.0-3.5％，磷酸二氢钾0.1-1.0％，氯化钠0.05-0.3％，酵母浸粉0.1-1.0％；
    2)种子培养：在无菌条件下从培养好的固体斜面上用接种铲划一满环长势良好的菌株接入无菌种子培养基中，该种子培养基初始pH值为5-8，在培养温度28-35℃，转速200-500rpm的条件下培养菌体到对数生长期；
    种子培养基配方按重量百分比包括如下成分：可溶性淀粉1.0-3.5％、磷酸二氢钾0.1-0.5％、尿素0.1-0.3％、硫酸镁0.05-0.1％、碳酸钙0.1-0.3％、酵母浸粉0.1-1.0％、玉米浆0.1-1.0％、硫酸铵0.1-0.6％、阿魏酸0.1-0.3％；
    3)发酵：将培养至对数生长期的种子液在无菌条件下以5-15％的体积比接入发酵培养基；发酵培养基初始pH值为7.2-7.8，在温度30-40℃，搅拌转速200-500rpm，通气量1:0.5的条件下发酵70-120h；发酵配方按重量百分比包括如下成分：
    可溶性淀粉2.0-5.0％、磷酸二氢钾0.1-0.3％、尿素0.1-0.5％、硫酸镁0.05-0.1％、碳酸钙0.5-2.0％、酵母浸粉0.1-1.0％、硫酸铵0.1-0.5％、阿魏酸1.0-3.0％。
  6. 如权利要求5所述的一种香兰素的生产方法，其特征在于，还包括如下步骤：
    4)提取：从发酵液中提取所需的香兰素。发酵结束，将发酵液升温至80℃，进行陶瓷膜过滤；然后滤液进行超滤去除小分子蛋白，色素，最后超滤液进行RO进行浓缩，浓缩5-10倍，将浓缩液PH值调至5-6，降温结晶，得香兰素粗品。
  7. 如权利要求6所述的一种香兰素的生产方法，其特征在于：步骤4)中陶瓷膜过滤为去除菌体和大分子蛋白。
  8. 如权利要求6所述的一种香兰素的生产方法，其特征在于：步骤4)的超滤为去除小分子蛋白和色素。
PCT/CN2016/070788 2015-07-27 2016-01-13 一种沙链霉菌的用途及香兰素的生产方法 WO2017016199A1 (zh)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1421523A (zh) * 2002-07-22 2003-06-04 江南大学 一种黑曲霉菌及用其生产香草酸和香草醛的微生物转化方法
CN101165168A (zh) * 2006-10-20 2008-04-23 上海爱普香料有限公司 一株链霉菌及利用其生物转化阿魏酸生产香兰素的方法
EP2157184A1 (en) * 2007-04-19 2010-02-24 Biokab, S.A. DE C.V. Process for producing vanilin from microorganisms immobilized by surface cuture
EP0885968B3 (en) * 1997-06-19 2012-10-17 Givaudan SA Process for the production of vanillin
CN105132472A (zh) * 2015-07-27 2015-12-09 厦门欧米克生物科技有限公司 一种沙链霉菌的用途及香兰素的生产方法

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20120028083A (ko) * 2010-09-14 2012-03-22 고려대학교 산학협력단 항균 활성을 갖는 스트렙토마이세스 프사모티쿠스 균주 및 이를 이용한 항생물질의 제조방법
CN104560825B (zh) * 2014-12-31 2017-10-27 中国科学院广州能源研究所 一种可在碱性条件下高效降解木质素的沙链霉菌及其应用

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0885968B3 (en) * 1997-06-19 2012-10-17 Givaudan SA Process for the production of vanillin
CN1421523A (zh) * 2002-07-22 2003-06-04 江南大学 一种黑曲霉菌及用其生产香草酸和香草醛的微生物转化方法
CN101165168A (zh) * 2006-10-20 2008-04-23 上海爱普香料有限公司 一株链霉菌及利用其生物转化阿魏酸生产香兰素的方法
EP2157184A1 (en) * 2007-04-19 2010-02-24 Biokab, S.A. DE C.V. Process for producing vanilin from microorganisms immobilized by surface cuture
CN105132472A (zh) * 2015-07-27 2015-12-09 厦门欧米克生物科技有限公司 一种沙链霉菌的用途及香兰素的生产方法

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