WO2018133399A1 - 一种利用微生物生产对羟基苯甲醛的方法 - Google Patents

一种利用微生物生产对羟基苯甲醛的方法 Download PDF

Info

Publication number
WO2018133399A1
WO2018133399A1 PCT/CN2017/097297 CN2017097297W WO2018133399A1 WO 2018133399 A1 WO2018133399 A1 WO 2018133399A1 CN 2017097297 W CN2017097297 W CN 2017097297W WO 2018133399 A1 WO2018133399 A1 WO 2018133399A1
Authority
WO
WIPO (PCT)
Prior art keywords
hydroxybenzaldehyde
fermentation
medium
strain
producing
Prior art date
Application number
PCT/CN2017/097297
Other languages
English (en)
French (fr)
Inventor
赵希景
邢晨光
黄志强
马芳
龚宏灿
Original Assignee
厦门欧米克生物科技有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 厦门欧米克生物科技有限公司 filed Critical 厦门欧米克生物科技有限公司
Priority to EP17893308.1A priority Critical patent/EP3483258B1/en
Priority to ES17893308T priority patent/ES2850774T3/es
Publication of WO2018133399A1 publication Critical patent/WO2018133399A1/zh

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/07—Bacillus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/24—Preparation of oxygen-containing organic compounds containing a carbonyl group
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/07—Bacillus
    • C12R2001/11—Bacillus megaterium

Definitions

  • the invention relates to the field of biotechnology, and in particular to a method for producing p-hydroxybenzaldehyde by using microorganisms.
  • P-hydroxybenzaldehyde having a molecular formula of C 7 H 6 O 2 , having a molecular weight of 122.12, a pale yellow or off-white crystal having a slight aromatic odor.
  • the main use is for important intermediates in the pharmaceutical and fragrance industries.
  • P-hydroxybenzaldehyde is a pale yellow or off-white crystalline substance with a slight aromatic odor, melting point: 112-116 ° C, relative density 1.129, boiling point: 246.6 ° C.
  • Parabens are important intermediates in the pharmaceutical and fragrance industries. It can be used in the pharmaceutical industry to synthesize amoxicillin (amoxicillin), antibacterial synergist trimethoprim (TMP), 3,4,5-trimethoxybenzaldehyde, p-hydroxyglycine, hydroxyaminobenzyl cephalosporin , artificial gastrodia, azadirachtin, escarol, etc.; used in the perfume industry for the synthesis of vanillin, ethyl vanillin, jasmonaldehyde, syringaldehyde, anisaldehyde and raspberry ketone and other spices.
  • the main object of the present invention is to provide a strain Bacillus megatherium OMK-11 having the production ability of p-hydroxybenzaldehyde, which is deposited under the China National Culture Collection as CCTCC NO: M2016658.
  • the preservation location is Wuhan City, Hubei province, Wuhan University: preservation time November 20, 2016.
  • Another object of the invention is to provide the use of said strain in the production of p-hydroxybenzaldehyde.
  • a further object of the present invention is to provide a process for producing p-hydroxybenzaldehyde which employs the aforementioned strain.
  • the invention provides a strain with p-hydroxybenzaldehyde production capacity, and uses the strain to ferment and produce p-hydroxybenzaldehyde, and the fermentation method produces p-hydroxybenzaldehyde by using natural raw materials such as saccharides and amino acids, and generates targets through microbial metabolism.
  • a method of product which belongs to low temperature and low pressure, relatively safe, simple operation and less pollution, and is a safe and environmentally friendly production method.
  • the product of the invention has a high output and is a production method with industrial prospects.
  • Figure 1 is a liquid phase detection spectrum of the L-tyrosine standard of Example 1;
  • Example 2 is a liquid phase detection spectrum of a p-hydroxybenzaldehyde standard in Example 1;
  • Example 3 is a liquid phase detection spectrum of a p-hydroxybenzaldehyde fermentation broth in Example 1.
  • a strain of Bacillus megatheriumOMK-11 with the production capacity of p-hydroxybenzaldehyde was isolated from the soil of ginger planting land. The strain was deposited with the China Center for Type Culture Collection on November 20, 2016, and the deposit number is CCTCC NO:M. 2016658.
  • the strain glucose, sucrose, ribose and arabinose can be used.
  • the 18s rRNA and 26s rRNA sequences of the strain are 99% similar to the 18s rRNA and 26s rRNA sequences of the Bacillus standard strain, and the strain is identified as Bacillus. . Rod-shaped, rounded at the end. Single or short chain arrangement. Gram positive.
  • strain activation, seed culture and fermentation of the strain can be carried out as follows:
  • strain activation under sterile conditions, a uniform solution of cyclamate from the glycerol tube is uniformly coated on the solid slant medium, and cultured in a biochemical incubator at 20-37 ° C for 24-48 h;
  • a well-grown strain is inserted into the sterile seed culture medium from the cultured solid slope with a inoculation shovel.
  • the seed culture medium has an initial pH of 5-8, and the culture temperature is 28-35 ° C, and the rotation speed is 150. Culture the cells to a logarithmic growth phase at -500 rpm;
  • the seed medium formula is as follows, according to the mass ratio: 1.0-3.5% of the saccharide raw material; 0.1-1.0% of the inorganic salt; 0.1-1.0% of the yeast dipping powder;
  • Fermentation the seed liquid cultured to the logarithmic growth phase is connected to the fermentation medium under a sterile condition at a volume ratio of 5-20%;
  • Fermentation medium initial pH 3.0-10.0, at a temperature of 30-40 ° C, stirring speed 200-500 rpm, fermentation under aeration conditions for 7-20h; then reduce ventilation, continue fermentation to 70-80h;
  • the fermentation medium comprises, by mass ratio, 2.0-5.0% of the saccharide raw material; 0.1-1.0% of the inorganic salt; 1.0-2.5% of the yeast dipping powder; and 1.0-3.0% of the tyrosine.
  • the solid slant medium is preferably a broth medium.
  • the saccharide raw material comprises at least one of glucose, sucrose and starch.
  • the inorganic salt includes a potassium salt or a sodium salt.
  • the step 3) initial pH of the fermentation medium is preferably 5.0-9.0.
  • a well-grown strain from a cultured solid slope was placed in a sterile seed culture medium at an initial pH of 5, and cultured at a culture temperature of 28 ° C and a rotation speed of 200 rpm. Bacterial to logarithmic growth phase.
  • the seed medium was formulated as follows, by mass ratio (%): glucose 1.0; NaCl 0.5; yeast soaked powder 0.1.
  • 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 fermentation medium was initially pH 5.0, fermented for 15 h at a temperature of 40 ° C, a stirring speed of 200 rpm, and an aeration of 1:0.1; then the ventilation was reduced to 1:0.01, and the fermentation was continued for 55 h.
  • the fermentation formula was as follows (%): glucose 1.0; NaCl 0.5; yeast dip 1.0; tyrosine 2.0; trace elements.
  • test results are shown in Figures 1 to 3.
  • the results of Figures 1 to 3 show that the fermentation broth contains p-hydroxybenzaldehyde.
  • a well-grown strain from a cultured solid slope was placed in a sterile seed culture medium at an initial pH of 6 and cultured at a culture temperature of 30 ° C and a rotation speed of 250 rpm. Bacterial to logarithmic growth phase.
  • the seed medium was formulated as follows (%): sucrose 3.5; KCl 0.5; yeast dip 1.0.
  • 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 fermentation medium was initially pH 9.0, fermented for 7 h at a temperature of 35 ° C, a stirring speed of 350 rpm, and an aeration of 1:0.2; then the ventilation was reduced to 1:0.02, and the fermentation was continued for 73 h.
  • the fermentation formula was as follows (%): sucrose 4.0; KCl 0.5; yeast immersion powder 1.0-2.5; tyrosine 1.0-3.0; trace elements.
  • a well-grown strain is inserted into the sterile seed culture medium from the cultured solid slope with a inoculating shovel.
  • the seed medium is initially pH8 and cultured at a culture temperature of 35 ° C and a rotation speed of 150 rpm. Bacterial to logarithmic growth phase.
  • the seed medium was formulated as follows (%): starch 3.5; NaCl 0.5; yeast dip 1.0.
  • the seed liquid cultured to the logarithmic growth phase was introduced into the fermentation medium under aseptic conditions at a volume ratio of 15%.
  • the fermentation medium was initially pH 7.0, fermented for 10 h at a temperature of 35 ° C, a stirring speed of 400 rpm, and an aeration of 1:0.1; then the air was lowered to 1:0.01, and the fermentation was continued for 65 hours.
  • the fermentation formula is as follows (%):
  • the seed medium was prepared, and the medium composition was as follows (g/L): glucose 10, potassium dihydrogen phosphate 7, sodium chloride 2.5, yeast dip powder 10.0, solvent water, initial pH 7.0, and sterilization at 121 ° C for 30 min.
  • the fermentation medium was prepared, and the medium composition was as follows (g/L): glucose 20, dipotassium hydrogen phosphate 4, yeast dip 1.0, tyrosine 10.0, trace element 100 ⁇ l, solvent water, initial pH 7.5. Sterilize at 121 ° C for 30 min.
  • Fermentation of natural p-hydroxybenzaldehyde 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 30 ° C, and the shaking speed is 200 rpm. The shaker was fermented for 20 hours to reduce the rotation speed of the shaker to 80 rpm, and then the fermentation was continued for 80 hours, and the concentration of natural p-hydroxybenzaldehyde in the fermentation broth was determined by HPLC to be 2.0 g/L.
  • the seed medium and the fermentation medium were prepared in the same manner as in Example 4.
  • Preparation of seeds Aseptic transfer of low temperature glycerol tube OKK-11 strain on fresh, sterile solid plate, activated at 28 ° C for 2 days, picking colonies inoculated in a 3L seed tank containing 1.8L seed medium for seed culture, ventilation The seeding liquid was obtained by stirring at 300 rpm and culturing at 30 ° C for 24 hours.
  • Fermentation of natural p-hydroxybenzaldehyde 10.2L of the prepared fermentation medium was charged into a 20L fermenter, sterilized at 121 °C for 30 min, and then the cultured 1.8 L seed solution was placed in a 20 L fermenter for fermentation. At 30 ° C, the stirring speed was 400 rpm, and the aeration ratio was 1:0.1. The fermentation was reduced to 1:0.01 by fermentation for 6 hours, and fermentation was continued for 54 hours. At the end of the fermentation, the concentration of natural p-hydroxybenzaldehyde in the fermentation broth was determined by HPLC to be 3 g/L.
  • the present invention provides a strain having a production ability of p-hydroxybenzaldehyde, and the strain is used for fermentation to produce p-hydroxybenzaldehyde.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Virology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Medicinal Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

一种利用微生物生产对羟基苯甲醛的方法，该微生物为具有对羟基苯甲醛生产能力的菌株Bacillus megatherium OMK-11，该菌株在中国典型培养物保藏中心的保藏号为CCTCC NO：M 2016658。采用微生物法生产，安全，且产品产量较高，是具有工业化前景的生产方法。

Description

一种利用微生物生产对羟基苯甲醛的方法 技术领域
本发明涉及生物技术领域，具体涉及一种利用微生物生产对羟基苯甲醛的方法。
背景技术
对羟基苯甲醛，分子式为C7H6O2，分子量为122.12，一种浅黄色或类白色结晶体，微有芳香气味。主要用途是用于医药工业和香料工业的重要中间体。
一、物理性质
对羟基苯甲醛是一种浅黄色或类白色结晶体，微有芳香气味，熔点：112～116℃，相对密度1.129，沸点：246.6℃。
易溶于乙醇、乙醚、丙酮、乙酸乙酯、DMF，稍溶于水(在30.5℃水中溶解度为1.388g/100mL)，溶于苯(在65℃苯中溶解度为3.68g/mL)。
二、用途
对羟基苯甲醛是医药工业和香料工业的重要中间体。在医药工业中可用于合成羟氨苄青霉素(阿莫西林)、抗菌增效剂甲氧苄胺嘧啶(TMP)、3，4，5-三甲氧基苯甲醛、对羟基甘氨酸、羟氨苄头孢霉素、人造天麻、杜鹃素、艾思洛儿等；在香料工业中用于合成香兰素、乙基香兰素、洋茉莉醛、丁香醛、茴香醛和复盆子酮等香料。
发明内容
本发明主要目的在于提供一种具有对羟基苯甲醛生产能力的菌株Bacillus megatherium OMK-11，该菌株在中国典型培养物保藏中心的保藏号为CCTCC NO：M2016658。保存地点为湖北省武汉市，武汉大学：保存时间2016年11月20日。
本发明的另一目的在于提供所述的菌株在生产对羟基苯甲醛的用途。
本发明的再一目的在于提供一种对羟基苯甲醛的生产方法，其采用前述的菌株。
本发明提供了一种具有对羟基苯甲醛生产能力的菌株，且利用该菌株进行发酵生产对羟基苯甲醛，发酵法生产对羟基苯甲醛是用天然原料糖质、氨基酸等，经过微生物代谢生成目标产物的一种方法，该方法属于低温低压，比较安全，操作简单，污染少，是一个安全环保的生产方法。本发明的产品产量较高，是一种具有工业化前景的生产方法。
附图说明
图1为实施例1中L-酪氨酸标准品的液相检测图谱；
图2为实施例1中对羟基苯甲醛标准品的液相检测图谱；
图3为实施例1中对羟基苯甲醛发酵液的液相检测图谱。
具体实施方式
菌种
从生姜种植地土壤中分离出一株具有对羟基苯甲醛生产能力的菌株Bacillus megatheriumOMK-11，该菌已于2016年11月20日保藏于中国典型培养物保藏中心，保藏号为CCTCC NO：M 2016658。
该菌株：可利用葡萄糖、蔗糖、核糖和阿拉伯糖等，该菌株的18s rRNA和26s rRNA序列与芽孢杆菌标准株的18s rRNA和26s rRNA序列有99％的相似性，该菌鉴定为芽孢杆菌属。杆状，末端圆。单个或呈短链排列。革兰氏阳性。
该菌株的菌种活化、种子培养和发酵可采用如下方法：
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20-37℃培养24-48h；
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pH5-8，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，按质量比包括：糖质原料1.0-3.5％；无机盐0.1-1.0％；酵母浸粉0.1-1.0％；
3)发酵：将培养至对数生长期的种子液在无菌条件下以5-20％的体积比接入发酵培养基；
发酵培养基初始pH3.0-10.0，在温度30-40℃，搅拌转速200-500rpm，通气条件下发酵7-20h；然后减小通风，继续发酵至70-80h；
发酵培养基，按质量比包括：糖质原料2.0-5.0％；无机盐0.1-1.0％；酵母浸粉1.0-2.5％；酪氨酸1.0-3.0％。
所述的固体斜面培养基优选为肉汤培养基。
所述的糖质原料，包括葡萄糖，蔗糖，淀粉中的至少一种。
所述的无机盐包括钾盐或钠盐。
所述的步骤3)发酵培养基初始pH优选为5.0-9.0。
实施例1
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pH5，在培养温度28℃，转速200rpm的条件下培养菌体到对数生长期。
种子培养基配方如下，按质量比(％)：葡萄糖1.0；NaCl 0.5；酵母浸粉0.1。
发酵工艺
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pH5.0，在温度40℃，搅拌转速200rpm，通气量1∶0.1的条件下发酵15h；然后降低通风至1∶0.01，继续发酵55h。
发酵配方如下(％)：葡萄糖1.0；NaCl 0.5；酵母浸粉1.0；酪氨酸2.0；微量元素。
发酵液检测方法：高效液相色谱法(HPLC法)
柱型：COSMOSIL；5C18-AR-II；4.6ID*250MM
柱温：30℃
流动相：甲醇与水(0.1％磷酸)比例为55∶45
流速：0.8mL/min，UV：216nm。
检测结果见图1至图3，图1至图3结果表明，发酵液中含有对羟基苯甲醛。
实施例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pH6，在培养温度30℃，转速250rpm的条件下培养菌体到对数生长期。
种子培养基配方如下(％)：蔗糖3.5；KCl 0.5；酵母浸粉1.0。
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 9.0，在温度35℃，搅拌转速350rpm，通气量1∶0.2的条件下发酵7h；然后减小通风至1∶0.02，继续发酵73h。
发酵配方如下(％)：蔗糖4.0；KCl 0.5；酵母浸粉1.0-2.5；酪氨酸1.0-3.0；微量元素。
实施例3
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pH8，在培养温度35℃，转速150rpm的条件下培养菌体到对数生长期。
种子培养基配方如下(％)：淀粉3.5；NaCl 0.5；酵母浸粉1.0。
发酵
将培养至对数生长期的种子液在无菌条件下以15％的体积比接入发酵培养基。发酵培养基初始pH7.0，在温度35℃，搅拌转速400rpm，通气量1∶0.1的条件下发酵10h；然后降低通风至1∶0.01，继续发酵65h。发酵配方如下(％)：
淀粉4.0；KCl 1.0；酵母浸粉2.5；酪氨酸3.0；微量元素。
实施例4
天然对羟基苯甲醛的生产(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10，磷酸二氢钾7，氯化钠2.5，酵母浸粉10.0，溶剂为水，初始pH7.0。121℃灭菌30min。
配制发酵培养基，其培养基组成如下(g/L)：葡萄糖20，磷酸氢二钾4，酵母浸粉1.0，酪氨酸10.0，微量元素100微升，溶剂为水，初始pH7.5。121℃灭菌30min。
种子的制备：无菌转移低温甘油管OMK-11菌株于新鲜、无菌固体平板上，28℃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24h，得到种子液。
天然对羟基苯甲醛的发酵：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30℃，摇床转速200rpm。摇瓶发酵20h把摇床转速降至80rpm，然后继续发酵80h后用HPLC法测定发酵液中天然对羟基苯甲醛的浓度为2.0g/L。
实施例5：天然对羟基苯甲醛的生产(生物反应器发酵)
种子培养基、发酵培养基配制同实施例4。
种子的制备：无菌转移低温甘油管OMK-11菌株于新鲜、无菌固体平板上，28℃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24h，得到种子液。
天然对羟基苯甲醛的发酵：将配制好的发酵培养基10.2L装入20L发酵罐中，121℃灭菌30min，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30℃，搅拌转速400rpm，通气比1∶0.1。发酵6h降低通风至1∶0.01，继续发酵54h。发酵结束用HPLC法测定发酵液中天然对羟基苯甲醛的浓度为3g/L。
工业实用性
本发明提供了一种具有对羟基苯甲醛生产能力的菌株，且利用该菌株进行发酵生产对羟基苯甲醛。

Claims (9)

  1. 具有对羟基苯甲醛生产能力的菌株Bacillus megatherium OMK-11，该菌株在中国典型培养物保藏中心的保藏号为CCTCC NO：M2016658。
  2. 如权利要求1所述的菌株在生产对羟基苯甲醛的用途。
  3. 一种对羟基苯甲醛的生产方法，其采用权利要求1所述的菌株。
  4. 如权利要求3所述的一种对羟基苯甲醛的生产方法，其特征在于，包括如下步骤：
    1)菌种活化：
    2)种子培养：
    在种子培养基中进行，培养至对数生长期；该种子培养基初始pH5-8，种子培养基按质量比包括：糖质原料1.0-3.5％；无机盐0.1-1.0％；酵母浸粉0.1-1.0％；
    3)发酵：
    在发酵培养基中进行，发酵培养基初始pH3.0-10.0，发酵培养基按质量比包括：糖质原料2.0-5.0％；无机盐0.1-1.0％；酵母浸粉1.0-2.5％；酪氨酸1.0-3.0％。
  5. 如权利要求4所述的一种对羟基苯甲醛的生产方法，其特征在于：
    1)所述的菌种活化：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20-37℃培养24-48h；
    2)所述的种子培养：
    该种子培养在培养温度28-35℃，转速150-500rpm的条件下培养菌体到对数生长期；
    3)所述的发酵步骤包括：将培养至对数生长期的种子液在无菌条件下以5-20％的体积比接入发酵培养基；
    发酵培养基在温度30-40℃，搅拌转速200-500rpm，通气条件下发酵7-20h；然后减小通风，继续发酵至50-100h。
  6. 如权利要求5所述的一种对羟基苯甲醛的生产方法，其特征在于，步 骤1)所述的固体斜面培养基为肉汤培养基。
  7. 如权利要求4或5所述的一种对羟基苯甲醛的生产方法，其特征在于，步骤2)和3)所述的糖质原料，包括葡萄糖，蔗糖，淀粉中的至少一种。
  8. 如权利要求4或5所述的一种对羟基苯甲醛的生产方法，其特征在于，步骤2)和3)所述的无机盐包括钾盐或钠盐。
  9. 如权利要求4或5所述的一种对羟基苯甲醛的生产方法，其特征在于，步骤3)发酵培养基初始pH5.0-9.0。
PCT/CN2017/097297 2017-01-20 2017-08-14 一种利用微生物生产对羟基苯甲醛的方法 WO2018133399A1 (zh)

Priority Applications (2)

Application Number Priority Date Filing Date Title
EP17893308.1A EP3483258B1 (en) 2017-01-20 2017-08-14 Method for producing p-hydroxybenzaldehyde using microorganism
ES17893308T ES2850774T3 (es) 2017-01-20 2017-08-14 Método para la producción de p-hidroxibenzaldehído por un microorganismo

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201710046221.6 2017-01-20
CN201710046221.6A CN107118973B (zh) 2017-01-20 2017-01-20 一种利用微生物生产对羟基苯甲醛的方法

Publications (1)

Publication Number Publication Date
WO2018133399A1 true WO2018133399A1 (zh) 2018-07-26

Family

ID=59717925

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2017/097297 WO2018133399A1 (zh) 2017-01-20 2017-08-14 一种利用微生物生产对羟基苯甲醛的方法

Country Status (4)

Country Link
EP (1) EP3483258B1 (zh)
CN (1) CN107118973B (zh)
ES (1) ES2850774T3 (zh)
WO (1) WO2018133399A1 (zh)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113881715A (zh) * 2021-09-15 2022-01-04 湖北工业大学 一种r-（+）-2-（4-羟基苯氧基）丙酸的生物合成方法
CN114250254A (zh) * 2021-12-20 2022-03-29 宁夏清研高分子新材料有限公司 一种对羟基苯甲酸的微生物合成方法

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108271781A (zh) * 2018-01-09 2018-07-13 华南农业大学 一种来源于假单胞菌的对羟基苯甲醛及其生产方法和应用
CN108315361B (zh) * 2018-04-20 2021-04-13 安徽大学 一种利用微生物合成对羟基苯甲醛的方法
CN112391418B (zh) * 2020-11-20 2022-02-18 厦门欧米克生物科技有限公司 一种覆盆子酮的工业化发酵生产方法

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001092539A2 (en) * 2000-06-01 2001-12-06 E.I. Dupont De Nemours And Company Method for the production of p-hydroxybenzoate in species of pseudomonas and agrobacterium
CN101074421A (zh) * 2006-05-19 2007-11-21 中国石油化工股份有限公司 一株巨大芽孢杆菌及其应用
CN106011002A (zh) * 2016-05-20 2016-10-12 东莞市保得生物工程有限公司 一种巨大芽孢杆菌t317及其菌剂和菌剂制备方法

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH06277078A (ja) * 1993-03-23 1994-10-04 Kawasaki Steel Corp パラヒドロキシベンズアルデヒドの製造方法
AU2002213028A1 (en) * 2000-09-30 2002-04-15 North Carolina State University Exbb-exbd-tonb gene cluster of pseudomonas putida dot-t1e conferring tolerance to aromatic compounds and resistance to antibiotics
CN102443551B (zh) * 2011-08-19 2013-08-07 甘肃省商业科技研究所 一种枯草芽孢杆菌及其生物转化阿魏酸生产香兰素的方法
KR20140022694A (ko) * 2012-08-14 2014-02-25 삼성전자주식회사 생물학적 방법으로 파라-히드록시벤조산을 제조하는 방법

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001092539A2 (en) * 2000-06-01 2001-12-06 E.I. Dupont De Nemours And Company Method for the production of p-hydroxybenzoate in species of pseudomonas and agrobacterium
CN101074421A (zh) * 2006-05-19 2007-11-21 中国石油化工股份有限公司 一株巨大芽孢杆菌及其应用
CN106011002A (zh) * 2016-05-20 2016-10-12 东莞市保得生物工程有限公司 一种巨大芽孢杆菌t317及其菌剂和菌剂制备方法

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
"HY96-2", vol. 34, 28 February 2008 (2008-02-28) *
EI-SHISHTAWY: "Solid fermentation of wheat bran for hydrolytic en- zymes production and saccharification content by a local isolate Bacillus megatherium", BMC BIOTECHNOLOGY, vol. 29, no. 1, 24 April 2014 (2014-04-24), pages 29, XP021181800 *
FITZPATRICK: "Characterization of YqjM, an Old Yellow Enzyme Ho log from Bacillus subtilis Involved in the Oxidative Stress Response", THE JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 278, no. 22, 26 March 2003 (2003-03-26), pages 19891 - 19897, XP002439308 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113881715A (zh) * 2021-09-15 2022-01-04 湖北工业大学 一种r-（+）-2-（4-羟基苯氧基）丙酸的生物合成方法
CN114250254A (zh) * 2021-12-20 2022-03-29 宁夏清研高分子新材料有限公司 一种对羟基苯甲酸的微生物合成方法
CN114250254B (zh) * 2021-12-20 2024-01-05 宁夏清研高分子新材料有限公司 一种对羟基苯甲酸的微生物合成方法

Also Published As

Publication number Publication date
EP3483258A1 (en) 2019-05-15
ES2850774T3 (es) 2021-08-31
CN107118973A (zh) 2017-09-01
EP3483258A4 (en) 2020-02-19
CN107118973B (zh) 2019-10-22
EP3483258B1 (en) 2021-01-13

Similar Documents

Publication Publication Date Title
WO2018133399A1 (zh) 一种利用微生物生产对羟基苯甲醛的方法
CN114806974B (zh) 一株盐单胞菌菌株及其应用
TWI537388B (zh) 一種利用高產量枯草桿菌突變株進行半固態發酵生產表面素之方法
WO2017016199A1 (zh) 一种沙链霉菌的用途及香兰素的生产方法
CN111072670B (zh) 一种二酮哌嗪类化合物及其制备方法和用途
CN109402017B (zh) 一株盐单胞菌100-16-2及盐单胞菌100-16-2制备聚3-羟基丁酸酯的方法
CN111172049A (zh) 一株毒黄素高产菌株及其应用
CN110577936A (zh) 一种漆酶高效降解四环类和磺胺类抗生素的方法
CN111434775A (zh) 一种发酵制备达托霉素的方法
CN104962491A (zh) 一种除草剂2,4-d的降解菌株及其生产的菌剂和应用
CN105802872B (zh) 一种荧光假单胞菌和生产吩嗪酰胺的方法及其应用
CN102559551A (zh) 一株施氏假单胞菌及其培养方法与应用
CN105754908B (zh) 一种铜绿假单胞菌菌株及其应用
WO2018133395A1 (zh) 一种利用微生物生产苯甲醛的方法
US3994781A (en) Process for the production of protein
CN110684668B (zh) 一种降低养殖过程中铅污染的盐生杜氏藻养殖方法
CN112760231B (zh) 一种用于低分子量普鲁兰多糖制备的培养体系及生产工艺
RU2747583C1 (ru) Способ получения (2r, 3s)-изолимонной кислоты из подсолнечного масла с помощью дрожжей yarrowia lipolytica
JP3796540B2 (ja) 黄色色素の製造方法
CN116058371A (zh) 水杨酸在抑制病原菌生长活性以及检测或者辅助检测病原菌毒性强弱中的应用
CN102559556B (zh) 一种提高喹乐霉素产量的发酵方法
RU2073712C1 (ru) Штамм бактерий azotobacter vinelandii (lipman) - продуцент экзополисахарида
EP3663391A1 (en) A strain for the production of natural tyrosol and a preparation method of natural tyrosol
CN117821299A (zh) 一株能够缓解玉米盐碱胁迫的植物根际促生菌pj-3及其应用
CN117821300A (zh) 一株能够缓解玉米盐碱胁迫的植物根际促生菌pj-2及其应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 17893308

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2017893308

Country of ref document: EP

Effective date: 20190207

NENP Non-entry into the national phase

Ref country code: DE