WO2018133395A1 - 一种利用微生物生产苯甲醛的方法 - Google Patents

一种利用微生物生产苯甲醛的方法 Download PDF

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WO2018133395A1
WO2018133395A1 PCT/CN2017/096860 CN2017096860W WO2018133395A1 WO 2018133395 A1 WO2018133395 A1 WO 2018133395A1 CN 2017096860 W CN2017096860 W CN 2017096860W WO 2018133395 A1 WO2018133395 A1 WO 2018133395A1
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fermentation
medium
strain
seed
benzaldehyde
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French (fr)
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邢晨光
赵希景
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 EP17892107.8A priority Critical patent/EP3483257B1/en
Publication of WO2018133395A1 publication Critical patent/WO2018133395A1/zh

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    • C—CHEMISTRY; METALLURGY
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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
    • C12N1/205—Bacterial isolates
    • 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
    • C12R2001/37—Proteus
    • 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

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  • the invention relates to the production of benzaldehyde, in particular to a method for producing benzaldehyde by using microorganisms.
  • Benzaldehyde is the simplest aromatic aldehyde, commonly known as bitter almond oil, also known as benzoic aldehyde, molecular formula: C 7 H 6 O, colorless to pale yellow volatile liquid. It has a bitter almond flavor and is aromatic when burned.
  • Benzaldehyde is widely present in the plant kingdom, especially in the Rosaceae plant, mainly in the form of glycosides in the stems, leaves or seeds of plants, such as amygdalin in bitter almonds.
  • Benzaldehyde is an important organic chemical raw material. Mainly used as raw materials for the production of lauric aldehyde, lauric acid, phenylacetaldehyde, benzyl benzoate. It is also an important intermediate for dyes, perfumes and pharmaceuticals.
  • Benzaldehyde a molecular formula of C 7 H 6 O, slightly soluble in water, miscible in ethanol, ether, benzene, chloroform.
  • the pure product is a colorless liquid
  • the industrial product is a colorless to pale yellow liquid, has a bitter almond smell, melting point: 28 ° C, boiling point: 178.1 ° C.
  • fragrance 2 can be used as a special top fragrance, used in floral formula, such as lilac, white orchid, jasmine, violet, acacia, sunflower, sweet bean, plum, orange blossom, etc. It can also be used in soap. It can also be used as a flavoring agent in almonds, berries, butter, cherries, coconuts, apricots, peaches, big walnuts, big plums, vanilla beans, and spices. Wine flavors such as rum, brandy, etc. are also used.
  • the main object of the present invention is to provide a strain Proteus vulgaris having a benzaldehyde production capacity.
  • Hauser OMK-9 the strain deposited in the China Center for Type Culture Collection is CCTCC NO: M2016671.
  • the preservation location is Wuhan City, Hubei province, Wuhan University: Saved on November 23, 2016.
  • Another object of the invention is to provide the use of said strain in the production of benzaldehyde.
  • Still another object of the present invention is to provide a process for producing benzaldehyde which employs the aforementioned strain.
  • the invention provides a strain having a benzaldehyde production capacity, and the strain is used for fermentation to produce benzaldehyde, and the fermentation method for producing benzaldehyde is a method for producing a target product through microbial metabolism by using natural raw materials such as saccharides and amino acids.
  • the process belongs to low temperature and low pressure, is relatively safe, simple in operation and less in 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.
  • Example 1 is a liquid phase detection spectrum of the L-phenylalanine standard in Example 1;
  • Example 2 is a liquid phase detection spectrum of the phenylacetic acid standard in Example 1;
  • Figure 3 is a liquid phase detection spectrum of the benzoic acid standard in Example 1;
  • Example 4 is a liquid phase detection spectrum of a benzaldehyde standard in Example 1;
  • Figure 5 is a liquid phase detection spectrum of the benzaldehyde fermentation broth of Example 1.
  • a strain of proteus vulgaris Hauser OMK-9 with benzaldehyde production capacity was isolated from the soil of the waste treatment station. The strain was deposited with the China Center for Type Culture Collection on November 23, 2016, and the deposit number is CCTCC NO: M2016671. .
  • the strain glucose, sucrose can be utilized.
  • the 18s rRNA and 26s rRNA sequences of this strain are 99% similar to the 18s rRNA and 26s rRNA sequences of the Proteus strain standard strain, which is identified as Proteus. It is a small bacillus which is negative for Gram stain, has no spores, is active and has rounded ends.
  • the activation, seed culture and fermentation of the strain can be carried out as follows:
  • 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;
  • the seed liquid cultured to the logarithmic growth phase is introduced into the fermentation medium under a sterile condition at a volume ratio of 5-20%;
  • the initial pH of the fermentation medium is 3.0-10.0, the temperature is 30-40 ° C, the stirring speed is 200-500 rpm, and the fermentation is carried out under aeration conditions for 7-20 h; then the ventilation is reduced, and the fermentation is continued for 50-100 h;
  • Fermentation medium by mass ratio: 2.0-5.0% of saccharide raw material; 0.1-1.0% of inorganic salt; 1.0-2.5% of yeast dipping powder; 1.0-3.0% of phenylalanine.
  • 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 of the well-preserved strain was inserted into the sterile seed culture medium from the cultured solid slope, and the seed culture medium was initially pH 6, at a culture temperature of 28 ° C and a rotation speed of 150 rpm. Culture the cells 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 1:0.2; then the ventilation was reduced to 1:0.02, and the fermentation was continued for 55 h.
  • the fermentation formula was as follows (%): glucose 5.0; NaCl 0.5; yeast dip 1.0; phenylalanine 2.0.
  • the test results are shown in Fig. 1 to Fig. 5.
  • the results of Fig. 1 to Fig. 5 indicate that the fermentation liquid contains benzaldehyde, and also contains by-products such as benzoic acid and phenylacetic acid.
  • 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 pH 5 at a culture temperature of 30 ° C and a rotation speed of 500 rpm. Culture the cells 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 300 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 dip powder 2.5; phenylalanine 1.5.
  • a well-grown strain of the well-preserved strain was inserted into the sterile seed culture medium from the cultured solid slope, and the seed culture medium was initially pH 7, at a culture temperature of 28 ° C and a rotation speed of 500 rpm. Culture the cells 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 1:0.2; then the ventilation was reduced to 1:0.02, and the fermentation was continued for 65 h.
  • the fermentation formula was as follows (%): starch 3.5; NaCl 0.5; yeast dip 1.0; phenylalanine 3.0.
  • the seed medium was prepared, and the medium composition was as follows (g/L): glucose 30, potassium dihydrogen phosphate 5, sodium chloride 2.5, yeast dip powder 10.0, solvent water, initial pH 7.0, sterilization at 121 ° C for 30 min.
  • the fermentation medium was prepared, and the medium composition was as follows (g/L): glucose 30, potassium dihydrogen phosphate 5, yeast dip powder 8, phenylalanine 10.0, solvent water, initial pH 7. Sterilization at 121 ° C for 30 min.
  • Fermentation of natural benzaldehyde 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 flask was fermented for 20 hours, the shaker speed was reduced to 80 rpm, and then the fermentation was continued for 80 hours, and the concentration of natural benzaldehyde in the fermentation broth was determined by HPLC to be 2.5 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-9 strain on fresh, sterile solid plate, activated at 28 °C for 2 days, pick 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 benzaldehyde 10.2L of the prepared fermentation medium was placed in a 20L fermenter, sterilized at 121 ° C for 30 min, and then the cultured 1.8 L seed solution was introduced into a 20 L fermenter for fermentation at a fermentation temperature of 30 ° C.
  • the stirring speed was 400 rpm and the aeration ratio was 1:0.2.
  • the fermentation was reduced to 1:0.02 by fermentation for 10 h, and fermentation was continued for 70 h.
  • the concentration of natural benzaldehyde in the fermentation broth was determined by HPLC to be 5 g/L.
  • the invention belongs to low temperature and low pressure, is relatively safe, has simple operation and less pollution.

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Abstract

一种利用微生物生产苯甲醛的方法，该微生物为具有苯甲醛生产能力的菌株Proteus vulgarisHauser OMK-9，该菌株在中国典型培养物保藏中心的保藏号为CCTCC NO：M2016671。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苯甲醛的方法 技术领域
本发明涉及苯甲醛的生产，特别涉及一种利用微生物生产苯甲醛的方法。
背景技术
苯甲醛是最简单的芳香醛，俗称苦杏仁油，又称为安息香醛，分子式：C7H6O，无色至浅黄色挥发性液体。有苦杏仁味，燃烧时有芳香味。苯甲醛广泛存在于植物界，特别是在蔷薇科植物中，主要以苷的形式存在于植物的茎皮、叶或种子中，例如苦杏仁中的苦杏仁苷。苯甲醛是一种重要的有机化工原料。主要用作生产月桂醛、月桂酸、苯乙醛、苯甲酸苄酯的原料。也是染料、香料及药品的重要中间体。
一、物理性质
苯甲醛，分子式为C7H6O，微溶于水，可混溶于乙醇、乙醚、苯、氯仿。纯品为无色液体，工业品为无色至淡黄色液体，有苦杏仁气味，熔点：28℃，沸点：178.1℃。饱和蒸气压(kPa)：0.13(26℃)。
二、用途
1、重要的化工原料，用于制月桂醛、月桂酸、苯乙醛和苯甲酸苄酯等，也用作香料。
2、可作为特殊的头香香料，微量用于花香配方，如紫丁香、白兰、茉莉、紫罗兰、金合欢、葵花、甜豆花、梅花、橙花等中。香皂中亦可用之。还可作为食用香料用于杏仁、浆果、奶油、樱桃、椰子、杏子、桃子、大胡桃、大李子、香荚兰豆、辛香等香精中。酒用香精如朗姆、白兰地等型中也用之。
3、医药、染料、香料的中间体。用于生产间氧基苯甲醛、月桂酸、月桂醛、品绿、苯甲酸苄酯、苄叉苯胺、苄叉丙酮等。用以调合皂用香精、食用香精等。
发明内容
本发明主要目的在于提供一种具有苯甲醛生产能力的菌株Proteus vulgaris  Hauser OMK-9，该菌株在中国典型培养物保藏中心的保藏号为CCTCC NO：M2016671。保存地点为湖北省武汉市，武汉大学：保存时间2016年11月23日。
本发明的另一目的在于提供所述的菌株在生产苯甲醛的用途。
本发明的再一目的在于提供一种苯甲醛的生产方法，其采用前述的菌株。
本发明提供了一种具有苯甲醛生产能力的菌株，且利用该菌株进行发酵生产苯甲醛，发酵法生产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。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-苯丙氨酸标准品的液相检测图谱；
图2为实施例1中苯乙酸标准品的液相检测图谱；
图3为实施例1中苯甲酸标准品的液相检测图谱；
图4为实施例1中苯甲醛标准品的液相检测图谱；
图5为实施例1中苯甲醛发酵液的液相检测图谱。
具体实施方式
菌种
从垃圾处理站土壤中分离出一株具有苯甲醛生产能力的菌株Proteus vulgaris Hauser OMK-9，该菌已于2016年11月23日保藏于中国典型培养物保藏中心，保藏号为CCTCC NO：M2016671。
该菌株：可利用葡萄糖、蔗糖。该菌株的18s rRNA和26s rRNA序列与变形杆菌标准株的18s rRNA和26s rRNA序列有99％的相似性，该菌鉴定为变形杆菌属。为革兰氏染色阴性、无芽胞、运动活泼、两端钝圆的小杆菌。
该菌株的活化、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25-30℃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：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50-100h；
发酵培养基，按质量比：糖质原料2.0-5.0％；无机盐0.1-1.0％；酵母浸粉1.0-2.5％；苯丙氨酸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pH 6，在培养温度28℃，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，按质量比(％)：葡萄糖1.0；NaCl 0.5；酵母浸粉0.1。
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pH5.0，在温度40℃，搅拌转速200rpm，通气量1∶0.2的条件下发酵15h；然后减小通风至1∶0.02，继续发酵55h。发酵配方如下(％)：葡萄糖5.0；NaCl 0.5；酵母浸粉1.0；苯丙氨酸2.0。
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5，图1至图5的结果表明发酵液中含有苯甲醛，同时还含有苯甲酸，苯乙酸等副产物。
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pH 5，在培养温度30℃，转速500rpm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300rpm，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2.5；苯丙氨酸1.5。
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pH 7，在培养温度28℃，转速500rpm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∶0.2的条件下发酵10h；然后减小通风至1∶0.02，继续发酵65h。发酵配方如下(％)：淀粉3.5；NaCl 0.5；酵母浸粉1.0；苯丙氨酸3.0。
实施例4
天然苯甲醛的生产(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30，磷酸二氢钾5，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30，磷酸二氢钾5，酵母浸粉8，苯丙氨酸10.0，溶剂为水，初始pH7。121℃灭菌30min。
种子的制备：无菌转移低温甘油管OMK-9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苯甲醛的浓度为2.5g/L。
实施例5：天然苯甲醛的生产(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-9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∶0.2。发酵10h降低通风至1∶0.02，继续发酵70h。发酵结束用HPLC法测定发酵液中天然苯甲醛的浓度为5g/L。
工业实用性
本发明属于低温低压，比较安全，操作简单，污染少。

Claims (9)

  1. 具有苯甲醛生产能力的菌株Proteus vulgaris Hauser OMK-9，其特征在于：该菌株在中国典型培养物保藏中心的保藏号为CCTCC NO：M2016671。
  2. 如权利要求1所述的菌株在生产苯甲醛的用途。
  3. 一种苯甲醛的生产方法，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pH 5-8；种子培养基按质量比：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：糖质原料2.0-5.0％；无机盐0.1-1.0％；酵母浸粉1.0-2.5％；苯丙氨酸1.0-3.0％。
  5. 如权利要求4所述的一种苯甲醛的生产方法，其特征在于，包括如下步骤：
    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25-30℃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50-100h；
  6. 如权利要求5所述的一种苯甲醛的生产方法，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。
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