WO2017008482A1 - 一株酵母菌及利用其发酵生产天然肉桂酸的方法 - Google Patents

一株酵母菌及利用其发酵生产天然肉桂酸的方法 Download PDF

Info

Publication number
WO2017008482A1
WO2017008482A1 PCT/CN2016/070787 CN2016070787W WO2017008482A1 WO 2017008482 A1 WO2017008482 A1 WO 2017008482A1 CN 2016070787 W CN2016070787 W CN 2016070787W WO 2017008482 A1 WO2017008482 A1 WO 2017008482A1
Authority
WO
WIPO (PCT)
Prior art keywords
fermentation
cinnamic acid
medium
yeast
cultured
Prior art date
Application number
PCT/CN2016/070787
Other languages
English (en)
French (fr)
Inventor
邢晨光
赵希景
黄志强
陈翠萍
刘伟
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 EP16823632.1A priority Critical patent/EP3299448B1/en
Priority to US15/574,767 priority patent/US10392672B2/en
Publication of WO2017008482A1 publication Critical patent/WO2017008482A1/zh

Links

Images

Classifications

    • 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
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/145—Fungal isolates
    • 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
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/16—Yeasts; Culture media therefor
    • 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
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • 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
    • C12R2001/645—Fungi ; Processes using fungi

Definitions

  • the present invention relates to a yeast and a method for producing natural cinnamic acid.
  • Cinnamic acid also known as ⁇ -phenylacrylic acid, 3-phenyl-2-oxic acid. White to light yellow powder with a molecular weight of 148.17. Micro with cinnamon aroma. Melting point, 133; boiling point, 300, density, 1.245; refractive index 1.555 (20 ° C), soluble in ethanol, methanol; soluble in benzene, acetic acid, petroleum ether, carbon disulfide and oil, slightly soluble in water. Natural cinnamic acid is mainly used in flavors and fragrances, food additives, pharmaceutical industry, beauty, etc.:
  • Flavor and fragrance It can be used as an aromatic mixture in soaps, shampoos, washing powders and daily cosmetics.
  • Cinnamic acid itself is a kind of spice, which has a good aroma retention effect. It is usually used as a fragrance raw material to make the aroma of the main fragrance more volatile.
  • Various esters of cinnamic acid can be used as a deodorant for beverages, cold drinks, candy, alcohol and other foods.
  • Cinnamic acid is synthesized by microbial enzymatic synthesis of L-phenylalanine.
  • L-phenylalanine is the main ingredient in the sweet food additive Aspartame.
  • food additives cinnamic acid microbial enzyme synthesis of L-phenylalanine.
  • L-phenylalanine is the main ingredient in the sweet food additive Aspartame.
  • United Unilever introduced the composition of cinnamic acid and pasteurization aid in WO 0187080 (2001, 11.22), which has strong sterilization and antiseptic effects.
  • the anti-mold and anti-corrosion sterilization using cinnamic acid can be applied to the preservation and anti-corrosion of food, vegetables and fruits.
  • Cinnamic acid is used in candied fruit to improve the taste of the taste, especially in food preservation and preservation, and has a pollution-free environmental preservative.
  • Alternatives sodium benzoate, potassium sorbate, etc.
  • wines can also be used in wines to make them brighter.
  • the pharmaceutical industry can be used for the synthesis of coronary heart disease important drugs lactic acid can be heart and heart pain, and synthetic chlorpheniric acid and cinnamyl piperazine, used to make "heart can be", local anesthetics, fungicides, Hemostatic drugs, etc. Chlorobenamic acid and cinnamyl piperazine can also be synthesized for use as a spine bone relaxant and an antispasmodic agent.
  • the casein amino acid enzyme is a key enzyme for melanin synthesis. It initiates the chain reaction of tyrosine to melanin biopolymer.
  • the cinnamic acid inhibits the formation of casein amino acids and has a certain inhibitory effect on ultraviolet light. It can make brown spots lighter or even disappear, and is one of the essential ingredients in advanced sunscreen.
  • the remarkable antioxidant effect of cinnamic acid has a good effect on slowing the appearance of wrinkles.
  • the cinnamic acid also has a good aroma retention effect, and is usually used as a flavoring ingredient in the flavoring agent.
  • the invention provides a yeast Rhodotorula sp.OMK-1, which is selected from the soil of a cinnamon plantation, and the bacteria has been preserved in the China Center for Type Culture Collection on May 27, 2015, and the deposit location is Hubei province. Wuhan University, Wuhan University, the deposit number is CCTCC M 2015326. Applicants have discovered that this yeast strain has the ability to produce natural cinnamic acid and applies it to the production of natural cinnamic acid.
  • a method for producing natural cinnamic acid characterized in that the production method is fermentation.
  • a method for producing natural cinnamic acid comprising the following steps:
  • the solid slant medium formulation comprises the following components: glucose 1.0-3.5%, potassium dihydrogen phosphate 0.1-1.0%, sodium chloride 0.05-0.3%, yeast dipping powder 0.1-1.0%;
  • 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: glucose 1.0-3.5%, potassium dihydrogen phosphate 0.1-1.0% urea 0.1-0.6% magnesium sulfate 0.05-0.5%, sodium chloride 0.05-0.3%, corn steep liquor 0.1-1.0%, urea 0.1-0.6%,
  • 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 6.8-7.2.0, at a temperature of 30- Fermentation at 40 ° C, stirring speed 200-500 rpm, aeration of 1:0.5, 70-100 h; fermentation formula includes the following components:
  • the step of step 4) is as follows: after the fermentation is finished, the fermentation liquid is heated, the bacteria are inactivated, and then the ceramic membrane is used to filter the bacteria, and the membrane filtrate is extracted with an organic solvent or a membrane filtrate. After concentration, the concentrate is directly acidified with dilute acid and crystallization at low temperature for separation and purification of natural cinnamic acid.
  • the elevated temperature is raised to 60-90 °C.
  • the invention adopts a fermentation method to produce natural cinnamic acid, adopts a natural raw material such as glucose, amino acid, etc., and a method for generating target product through microbial metabolism, the process belongs to low temperature and low pressure, relatively safe, simple operation and less pollution, and is a safe and environmentally friendly production. method.
  • Figure 1 is an HPLC chart in Example 3, demonstrating that the resulting product is natural cinnamic acid
  • Rhodotorula sp.OMK-1 A strain of Rhodotorula sp.OMK-1 with natural cinnamic acid production capacity was isolated from the soil under the cinnamon plantation in Haikou City, Hainan province, China. The strain was deposited with the China Center for Type Culture Collection on May 27, 2015. The deposit is located in Wuhan City, Hubei province, Wuhan University, and the deposit number is CCTCC M 2015326.
  • the cell is oval in shape, the colony is orange-yellow, round, the edges are neat, and the surface is smooth.
  • Glucose, sucrose, ribose and arabinose can be utilized and cannot be cultured with L-sorbitol, maltitol and erythritol.
  • the 18s rRNA and 26s rRNA sequences of this strain are 99% similar to the 18s rRNA and 26s rRNA sequences of the red yeast standard strain, which is identified as Rhodobacter.
  • the solid slant medium formulation is as follows: (%)
  • a well-grown strain is inserted into the sterile seed culture medium from the cultured solid slope by using a inoculating shovel.
  • the initial pH of the seed medium is 5-8, and the culture temperature is 28-35 °C.
  • the cells were cultured to a logarithmic growth phase at a rotational speed of 150 to 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 7.0, and the fermentation was carried out for 70 to 120 hours at a temperature of 30 to 40 ° C, a stirring speed of 200 to 500 rpm, and an aeration rate of 1:0.5.
  • the fermentation formula is as follows: (%)
  • the fermentation liquid is heated to 80 ° C to inactivate the bacteria, and then the ceramic membrane is used to filter the bacteria, the membrane filtrate is extracted with an organic solvent (such as ethyl acetate) or the membrane filtrate is directly acidified by dilute acid crystallization. Separation and purification of cinnamic acid.
  • an organic solvent such as ethyl acetate
  • Example 1 Production of natural cinnamic acid (triangular shake fermentation)
  • 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, phenylalanine 30, solvent is water
  • the initial pH was 7. Sterilization at 121 ° C for 30 min.
  • Fermentation of natural cinnamic acid 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. After the fermentation for 100 h, the concentration of natural cinnamic acid in the fermentation broth was determined by HPLC to be 15 g/L.
  • Example 2 Production of natural cinnamic acid (stirred reactor fermentation)
  • the seed medium and the fermentation medium were prepared in the same manner as in Example 1.
  • Fermentation of natural cinnamic acid 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 placed in 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 cycle is 110h.
  • the concentration of natural cinnamic acid in the fermentation broth was determined by HPLC to be 20 g/L.
  • Example 1 Take 50 mL of the fermentation broth in Example 1, and warm the mixture to 80 ° C in a water bath for 30 minutes to inactivate the cells.
  • the fermentation broth was centrifuged at 5000 rpm to remove the cells, then the pH was adjusted to 3 to 5 with dilute acid, static for 5 min, extracted with 50 mL of ethyl acetate, and the layers were separated.
  • the organic phase was suspended with a rotary evaporator to obtain a natural cinnamic acid product. 0.7g.
  • the HPLC chart is shown in Figure 1.
  • Example 2 The 12 L fermentation broth of Example 2 was taken, and the ceramic membrane was filtered at 60 ° C to collect 10 L of the filtrate. The filtrate was adjusted to pH 3 to 5 with dilute acid, and the acidified membrane filtrate was allowed to stand for 4 h, centrifuged, and dried to obtain 216 g of natural cinnamic acid.
  • the invention adopts the fermentation method to produce natural cinnamic acid, has low temperature and low pressure, is relatively safe, and has simple operation.

Landscapes

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

Abstract

提供了一种红酵母（Rhodotorula sp.）菌株OMK-1，其保藏号为CCTCC M 2015326。还提供了利用该红酵母菌株OMK-1发酵生产天然肉桂酸的方法，其包括菌种活化、种子培养、发酵和提取等步骤。

Description

一株酵母菌及利用其发酵生产天然肉桂酸的方法 技术领域
本发明涉及一种酵母菌以及天然肉桂酸的生产方法。
背景技术
肉桂酸，又名β‐苯丙烯酸、3‐苯基‐2‐丙烯酸。白色至淡黄色粉末，分子量148.17。微有桂皮香气。熔点，133；沸点，300，密度，1.245；折射率1.555(20℃)，溶于乙醇、甲醇；易溶于苯、醋酸、石油醚、二硫化碳及油类，微溶于水。天然肉桂酸主要用于香精香料、食品添加剂、医药工业、美容等方面：
1、香精香料：可作为芳香混合物，用于香皂、香波、洗衣粉、日用化妆品中。
肉桂酸本身就是一种香料，具有很好的保香作用，通常作为配香原料，可使主香料的香气更加清香挥发。肉桂酸的各种酯(如甲、乙、丙、丁等)都可用作定香剂，用于饮料、冷饮、糖果、酒类等食品。
肉桂酸用微生物酶法合成L‐苯丙氨酸。L‐苯丙氨酸是重要的食品添加剂—甜味阿斯巴甜(Aspartame)的主要原料。
2、食品添加剂：肉桂酸用微生物酶法合成L‐苯丙氨酸。L‐苯丙氨酸是重要的食品添加剂—甜味阿斯巴甜(Aspartame)的主要原料。英国联合利华在WO 0187080(2001，11.22)中介绍了肉桂酸和巴氏杀菌助剂组成，具有很强的杀菌、防腐作用。利用肉桂酸的防霉防腐杀菌可应用于粮食、蔬菜、水果中的保鲜、防腐。肉桂酸用于蜜饯中，能改善口感风味，尤其是在食品防腐保鲜上，具有无公害的环保防腐剂。替代(苯甲酸钠，山梨酸钾，等产品)还可用在葡萄酒中，使其色泽光鲜。
3、医药工业：可用于合成治疗冠心病的重要药物乳酸可心定和心痛平，及合成氯苯氨丁酸和肉桂苯哌嗪，用来制造“心可安”，局部麻醉剂、杀菌剂、止血药等。还可合成氯苯氨丁酸和肉桂苯哌嗪，用作脊锥骨骼松弛剂和镇痉剂。主 要用于脑血栓，脑动脉硬化，冠状动脉硬化等病症。对肺腺癌细胞增殖有明显抑制作用。肉桂酸是A－5491人肺腺癌细胞有效的抑制剂，在抗癌方面具有极大的应用价值。
4、美容方面：酪氨基酸酶是黑色素合成关键酶，它启动了由酪氨酸转化为黑色素生物聚合体的级链反应，肉桂酸有抑制形成酪氨基酸酶的作用，对紫外线有一定的隔绝作用，能使褐斑变浅，甚至消失，是高级防晒霜中必不可少的成分之一。肉桂酸显著的抗氧化功效对于减慢皱纹的出现有很好的疗效。肉桂酸同时还具有很好的保香作用，通常作为配香原料，被用作日化香精中的定香剂。
合成肉桂酸的方法众多，主要合成方法如下：1、Perkin合成法；2、苯甲醛‐丙酮法；3、苄叉二氯‐无水醋酸钠法。这些方法或流程长，温度高，能耗大，收率低；或副产物多，分离纯化难，污染严重。4、肉桂醛氧化为肉桂酸法，以H2O2(浓度要求为90％～100％属危险品)、NaClO2等无机氧化物为氧化剂进行氧化，需要大量有机溶剂如丙腈、苯等，污染环境，不利于工业化。
发明内容
本发明的目的在于提供一种酵母菌的用途以及天然肉桂酸的生产方法，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：
本发明提供一种酵母菌Rhodotorula sp.OMK-1，其是从肉桂种植园土壤中筛选出来的，该菌已于2015年5月27日保藏于中国典型培养物保藏中心，保藏地点为湖北省武汉市，武汉大学，保藏号为CCTCC M 2015326。申请人发现该酵母菌菌株具有生产天然肉桂酸的能力，并将其应用于天然肉桂酸的生产。
前述的一种酵母菌Rhodotorula sp.OMK-1的用途，其用于生产天然肉桂酸。
一种天然肉桂酸的生产方法，其特征在于：所述的生产方法为发酵。
一种天然肉桂酸的生产方法，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24-48小时；其中，所述的菌液为酵母菌Rhodotorula sp.OMK-1；
固体斜面培养基配方包括如下成分：葡萄糖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：葡萄糖1.0-3.5％、磷酸二氢钾0.1-1.0％尿素0.1-0.6％硫酸镁0.05-0.5％、氯化钠0.05-0.3％、玉米浆0.1-1.0％、尿素0.1-0.6％、
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6.8-7.2.0，在温度30-40℃，搅拌转速200-500rpm，通气量1:0.5的条件下发酵70-100h；发酵配方包括如下组分：
葡萄糖2.0-5.0％、磷酸二氢钾0.1-0.5％、尿素0.1-0.5％、硫酸镁0.05-0.5％、氯化钠0.05-3％、酵母浸粉0.1-1.0％、硫酸铵0.1-0.5％、苯丙氨酸1.0-2.0％。
4)提取：从发酵液中提取所需的天然肉桂酸。
在本发明的较佳实施例中，步骤4)的步骤如下：发酵结束后，发酵液升温，把菌灭活，然后进行陶瓷膜过滤除菌体，膜过滤液用有机溶剂萃取或膜过滤液经过浓缩，然后浓缩液直接用稀酸酸化、低温结晶进行天然肉桂酸分离提纯。
在本发明的较佳实施例中，所述的升温为升至60-90℃。
本发明采用发酵法生产天然肉桂酸，采用天然原料葡萄糖、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。
附图说明
图1为实施例3中的HPLC图，证明所得的产物为天然肉桂酸
具体实施方式
1、菌种
从中国海南省海口市肉桂种植园树林下土壤中分离出一株具有天然肉桂酸生产能力的菌株Rhodotorula sp.OMK-1，该菌已于2015年5月27日保藏于中国典型培养物保藏中心，保藏地点为湖北省武汉市，武汉大学，保藏号为CCTCC M 2015326
该菌细胞卵形，菌落橘黄色，圆形，边缘整齐，表面光滑。可利用葡萄糖、蔗糖、核糖和阿拉伯糖，不能利用L‐山梨醇、麦芽糖醇和赤藻糖醇培养。该菌株的18s rRNA和26s rRNA序列与红酵母标准株的18s rRNA和26s rRNA序列有99％的相似性，该菌鉴定为红酵母属。
2、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度培养24～48小时。固体斜面培养基配方如下：(％)
葡萄糖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；
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：(％)
葡萄糖1.0-3.5、磷酸二氢钾0.1-1.0、尿素0.1-0.6、硫酸镁0.05-0.5、氯化钠0.05-0.3、玉米浆0.1-1.0和尿素0.1-0.6
4、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PH值为7.0，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：(％)
葡萄糖2.0-5.0、磷酸二氢钾0.1-0.5、尿素0.1-0.5、硫酸镁0.05-0.5、氯化钠0.05-3酵母浸粉0.1-1.0、硫酸铵0.1-0.5、苯丙氨酸1.0-2.0。
5、提取工艺
发酵结束后，发酵液升温至80℃把菌灭活，然后进行陶瓷膜过滤除菌体，膜过滤液用有机溶剂(如乙酸乙酯)萃取或膜过滤液直接用稀酸酸化结晶等方式进行桂酸分离提纯。
实施例1：天然肉桂酸的生产(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，尿素4，硫酸镁0.8，氯化钠2.5，玉米浆5，尿素6，溶剂为水，初始PH值为7.0。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，尿素5，硫酸镁0.6，酵母浸粉8，硫酸铵3，苯丙氨酸30，溶剂为水，初始pH值为7。121℃灭菌30min。
种子的制备：无菌转移低温甘油管Rhodotorula sp.OMK-1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。
天然肉桂酸的发酵：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。发酵100h后用HPLC法测定发酵液中天然肉桂酸的浓度为15g/L。
实施例2：天然肉桂酸的生产(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Rhodotorula sp.OMK-1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。
天然肉桂酸的发酵：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。发酵周期110h。发酵结束用HPLC法测定发酵液中天然肉桂酸的浓度为20g/L。
实施例3：天然肉桂酸的提取
取实施案例1中的50mL发酵液，水浴升温至80℃，保温30min，灭活菌体。发酵液进行5000rpm离心出去菌体，然后用稀酸调节PH至3～5，静止5min，加50mL乙酸乙酯萃取，分层，有机相用旋转蒸发仪把乙酸乙酯悬干得天然肉桂酸成品0.7g。其HPLC图见图1。
实施例4：天然肉桂酸的提取
取实施例2中12L发酵液，60℃进行陶瓷膜膜过滤，收集滤液10L。滤液用稀酸调PH至3～5，酸化后的膜过滤液静置4h，离心，干燥，最终得到天然肉桂酸216g。
工业实用性
本发明采用发酵法生产天然肉桂酸，低温低压，比较安全，操作简单。

Claims (6)

  1. 一种酵母菌Rhodotorula sp.OMK‐1，其特征在于：其保藏编号为CCTCC M2015326。
  2. 如权利要求1所述的一种酵母菌Rhodotorula sp.OMK‐1，其用于生产天然肉桂酸。
  3. 一种如权利要求2所述天然肉桂酸的生产方法，其特征在于：所述的生产方法为微生物发酵法。
  4. 一种肉桂酸的生产方法，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24-48小时；其中，所述的菌液为酵母菌Rhodotorula sp.OMK-1；
    固体斜面培养基配方包括如下成分：葡萄糖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：葡萄糖1.0-3.5％、磷酸二氢钾0.1-1.0％尿素0.1-0.6％硫酸镁0.05-0.5％、氯化钠0.05-0.3％、玉米浆0.1-1.0％、尿素0.1-0.6％；
    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6.8-7.2，在温度30-40℃，搅拌转速200-500rpm，通气量1:0.5的条件下发酵70-100h；发酵配方包括如下组分：
    葡萄糖2.0-5.0％、磷酸二氢钾0.1-0.5％、尿素0.1-0.5％、硫酸镁0.05-0.5％、氯化钠0.05-3％、酵母浸粉0.1-1.0％、硫酸铵0.1-0.5％、苯丙氨酸1.0-2.0％；
    4)提取：从发酵液中提取所需的天然肉桂酸。
  5. 如权利要求4所述的一种天然肉桂酸的生产方法，其特征在于，步骤4)的步骤如下：发酵结束后，发酵液升温，把菌灭活，然后进行陶瓷膜过滤除菌体，膜过滤液用有机溶剂萃取或膜过滤液经过浓缩，然后浓缩液直接用稀酸酸化、低 温结晶进行天然肉桂酸分离提纯。
  6. 如权利要求5所述的一种天然肉桂酸的生产方法，其特征在于：所述的升温为升至60-90℃。
PCT/CN2016/070787 2015-07-14 2016-01-13 一株酵母菌及利用其发酵生产天然肉桂酸的方法 WO2017008482A1 (zh)

Priority Applications (2)

Application Number Priority Date Filing Date Title
EP16823632.1A EP3299448B1 (en) 2015-07-14 2016-01-13 Yeast strain and production method of natural cinnamic acid using fermentation thereof
US15/574,767 US10392672B2 (en) 2015-07-14 2016-01-13 Yeast strain and method for producing natural cinnamic acid using the yeast strain for fermentation

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201510411874.0 2015-07-14
CN201510411874.0A CN105087387B (zh) 2015-07-14 2015-07-14 一株酵母菌及利用其发酵生产天然肉桂酸的方法

Publications (1)

Publication Number Publication Date
WO2017008482A1 true WO2017008482A1 (zh) 2017-01-19

Family

ID=54568725

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2016/070787 WO2017008482A1 (zh) 2015-07-14 2016-01-13 一株酵母菌及利用其发酵生产天然肉桂酸的方法

Country Status (4)

Country Link
US (1) US10392672B2 (zh)
EP (1) EP3299448B1 (zh)
CN (1) CN105087387B (zh)
WO (1) WO2017008482A1 (zh)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105087387B (zh) * 2015-07-14 2018-10-26 厦门欧米克生物科技有限公司 一株酵母菌及利用其发酵生产天然肉桂酸的方法

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS62248493A (ja) * 1986-04-21 1987-10-29 Idemitsu Kosan Co Ltd 桂皮酸の製造方法
CN104131017A (zh) * 2014-07-28 2014-11-05 江南大学 一种粘红酵母苯丙氨酸脱氨酶基因及其应用
CN105087387A (zh) * 2015-07-14 2015-11-25 厦门欧米克生物科技有限公司 一株酵母菌及利用其发酵生产天然肉桂酸的方法

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7105326B2 (en) * 2001-05-04 2006-09-12 E. I. Du Pont De Nemours And Company Methods for the production of tyrosine, cinnamic acid and para-hydroxycinnamic acid

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS62248493A (ja) * 1986-04-21 1987-10-29 Idemitsu Kosan Co Ltd 桂皮酸の製造方法
CN104131017A (zh) * 2014-07-28 2014-11-05 江南大学 一种粘红酵母苯丙氨酸脱氨酶基因及其应用
CN105087387A (zh) * 2015-07-14 2015-11-25 厦门欧米克生物科技有限公司 一株酵母菌及利用其发酵生产天然肉桂酸的方法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
LIANG, JINGLONG ET AL.: "Construction of Genetically Engineering Escherichia coli for Cinnamic Acid Production", ACADEMIC CONFERENCE ON ''NEW TECHNOLOGY AND NEW DEVELOPMENT OF FOOD INDUSTRY'' AND THE PROCEDDINGS OF THE ANNUAL CONFERENCE OF GUANGDONG INSTITUTE OF FOOD SCIENCE AND TECHNOLOGY IN 2014, 30 November 2014 (2014-11-30), pages 29 - 31, XP009508142 *

Also Published As

Publication number Publication date
US20180148802A1 (en) 2018-05-31
EP3299448A1 (en) 2018-03-28
US10392672B2 (en) 2019-08-27
CN105087387A (zh) 2015-11-25
EP3299448B1 (en) 2020-03-11
CN105087387B (zh) 2018-10-26
EP3299448A4 (en) 2018-12-19

Similar Documents

Publication Publication Date Title
CN101317646B (zh) 一种酸菜直投式发酵剂
JP6132398B2 (ja) エクオール以外のイソフラボン類の含有量が低いエクオール含有組成物
KR20160147148A (ko) 밀리타리스 동충하초버섯의 재배방법
CN103005382B (zh) 降胆固醇菌粉，含有该菌粉的降胆固醇复合调味酱及其应用
WO2017016199A1 (zh) 一种沙链霉菌的用途及香兰素的生产方法
WO2017008482A1 (zh) 一株酵母菌及利用其发酵生产天然肉桂酸的方法
CN115715283A (zh) 在丝状真菌中生产茉莉酸类
CN102090620A (zh) 具有抑菌活性的竹荪子实体提取物及其应用
CN106942692A (zh) 一种提高酱油酯香品质的方法及由其得到的制品
CN103014067B (zh) 发酵法生产水溶性姜黄色素的方法
KR101911640B1 (ko) 가스발생량이 낮은 류코노스톡 메센테로이드 cjlm627 균주 및 이를 이용한 김치의 제조방법
CN102334667B (zh) 一种发酵型奶味香料及其制备方法
CN102115768B (zh) 微生物同步发酵正十六烷生产十六碳二元酸的方法
KR20160030754A (ko) 유용미생물을 이용한 발효 커피 및 이의 제조 방법
KR101750288B1 (ko) 귤응애 방제용 조성물 및 그 방제 방법
CN105077465A (zh) 基于热爆预处理制备纯黑木耳或纯海带发酵液制品的方法
KR20020093690A (ko) 벌꿀음료 제조
WO2018133395A1 (zh) 一种利用微生物生产苯甲醛的方法
CN113907242B (zh) 一种包括罗汉果酸甲衍生物的食品防腐组合物及其制备方法
WO2020203966A1 (ja) エラスターゼ活性抑制剤、エラスターゼ活性抑制外用剤、及びエラスターゼ活性抑制飲食用組成物
KR100717611B1 (ko) 유산균을 이용한 발효 강황 조성물 및 그 제조방법
JP2007267662A (ja) 色落ち海苔加工食品素材の製造方法および海苔加工食品素材
KR20060105698A (ko) 감 껍질을 이용한 식초의 제조방법
JPS5937978B2 (ja) 食品または食品加工製品もしくは各種薬剤に添加するに適する消臭剤の製法
KR101806519B1 (ko) 발효식품의 바이오제닉 아민 저감화 방법

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: 16823632

Country of ref document: EP

Kind code of ref document: A1

WWE Wipo information: entry into national phase

Ref document number: 15574767

Country of ref document: US

NENP Non-entry into the national phase

Ref country code: DE

WWE Wipo information: entry into national phase

Ref document number: 2016823632

Country of ref document: EP