CN101857844B - Wine brewing yeast strain capable of generating 2-phenethyl alcohol by biotransformation of L-phenylanine - Google Patents
Wine brewing yeast strain capable of generating 2-phenethyl alcohol by biotransformation of L-phenylanine Download PDFInfo
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Abstract
The invention discloses a wine brewing yeast strain capable of generating 2-phenethyl alcohol by biotransformation of L-phenylanine, which is named Saccharomyces cerevisiae and was preserved in China Typical Culture Preservation Center on March 1st, 2009 with a preservation number of CCTCC M 29035. The bacterial strain is separated from soil, has an oval or ovate shape with a diameter of 5 to 10 micrometers and is yellowish. The bacterial strain has the typical physiological and biochemical characteristics of wine brewing yeasts. The 18S rDNA sequence of the bacterial strain is 99 percent similar to that of other wine brewing yeast strains. The bacterial strain can be used for producing natural 2-phenethyl alcohol by biotransformation of L-phenylanine. The production of the 2-phenethyl alcohol by a biotransformation method has the advantages of mild reaction conditions, green and natural products, low cost, environmental friendliness and the like.
Description
Technical field
The invention belongs to biological technical field, relate in particular to the new isolating yeast saccharomyces cerevisiae of a strain and be used for the method for carrying out bioconversion on L-phenylalanine to produce 2-phenethyl alcohol.
Background technology
2 phenylethyl alcohol has another name called bata-phenethyl alcohol, English 2-phenylethanol by name, and molecular formula is C8H10O, is water white liquid under the normal temperature, natural being present in some flowers and the essential oil of plant such as rose, jasmine etc.The rose scent that 2 phenylethyl alcohol has is popular to people deeply, is the basal component of all rose scent fragrance, and it is widely used in food, makeup, daily necessities, tobacco and medicine.
The working method of 2 phenylethyl alcohol can be divided into biological synthesis process, directly three kinds of extraction method and chemical synthesiss from plant.
Discover that yeast can start anew to synthesize, also can transform the L-phenylalanine(Phe) and generate 2 phenylethyl alcohol through amino acid decomposition approach.Kluyveromyces marxianus (Kluyveromyces marxianus), fermentation pichia spp (Pichia fermentans) and a certain amount of 2 phenylethyl alcohol of de novo synthesis in process of growth such as yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) and unusual debaryomyces hansenii (Hansenula anomala).Ehrlich in 1907 finds in some yeast cell, the L-phenylalanine(Phe) through transamination generate phenyl-pyruvic acid, decarboxylation forms phenylacetic aldehyde again, phenylacetic aldehyde is through oxydehydrogenation enzyme effect generation 2 phenylethyl alcohol, this approach is afterwards with the naming of Ehrlich.Utilizing this approach is the main method that present biological synthesis process prepares 2 phenylethyl alcohol through carrying out bioconversion on L-phenylalanine to produce 2-phenethyl alcohol.
The unusual debaryomyces hansenii of humans such as Albertazzi (Hansenula anomala CBS 110) does not contain containing 2 grams per liter L-phenylalanine(Phe)s to cultivate in the substratum of other nitrogenous sources and obtained 1.7 grams per liter 2 phenylethyl alcohols in 24 hours, and the same terms uses yeast saccharomyces cerevisiae (Saccharomyces cerevisiae NCYC 739) to obtain 1.5 grams per liter 2 phenylethyl alcohols (1994) down.Humans such as Huang fermentation pichia spp (Pichia fermentans L-5) transforms the L-phenylalanine(Phe) can obtain 453 mg/litre after 16 days 2 phenylethyl alcohol (2000).It is starting strain that people such as Stark adopt yeast saccharomyces cerevisiae (Saccharomyces cerevisiae Giv 2009), concentrates conversion 48 as a child to obtain 2.35 grams per liter 2 phenylethyl alcohols in the cultivation that contains 6 grams per liter L-phenylalanine(Phe)s.In view of the toxic action of 2 phenylethyl alcohol to yeast cell, make the output of 2 phenylethyl alcohol rest on a lower level, the researchist has adopted original position product stripping technique to improve the productive rate of 2 phenylethyl alcohol.People such as Stark, adopt yeast saccharomyces cerevisiae (Saccharomyces cerevisiae Giv 2009) to carry out two phase fed-batch and transform as extraction agent with oleyl alcohol, and final 2 phenylethyl alcohol concentration can reach 12.6 grams per liters.Though adopt this method 2 phenylethyl alcohol output to obtain bigger raising, brought a lot of difficulties for simultaneously the separation and Extraction of product, also increased production cost.
The method of from plant, extracting 2 phenylethyl alcohol is limited because of raw material sources, and the content such as 2 phenylethyl alcohol in the rose is very low in the raw material, and extraction cost is too high, and the product that obtains with this method only is applied in the high-grade spices at present; The environmental issue that the chemical synthesis ubiquity is severe, reaction conditions is generally more violent, and is higher to equipment requirements, and product is not natural; Biological synthesis process has that Product Green is natural, cost is low, reaction conditions is gentle, little or the like the outstanding advantage of environmental pollution, is the direction of 2 phenylethyl alcohol preparation research in the future, and fabulous development prospect is arranged.But producing bacterial strain from occurring in nature screening 2 phenylethyl alcohol is a very large order; Wild strain output that screens and productive rate are often very low; Tolerance to the product 2 phenylethyl alcohol is low; So produce the technology prematurity still of 2 phenylethyl alcohol in the world with biological synthesis process, the report that does not also have industrialization to produce at present.
Summary of the invention
The objective of the invention is to the needs of existing market the natural green product; And biological process produce the 2 phenylethyl alcohol production concentration low, not have problems such as production bacterial strain of getting well; The yeast saccharomyces cerevisiae of the new isolating production 2 phenylethyl alcohol of one strain is provided, and the method with this bacterial strain carrying out bioconversion on L-phenylalanine to produce 2-phenethyl alcohol is provided.
The yeast saccharomyces cerevisiae that the present invention relates to is called Saccharomyces cerevisiae P-3, is preserved in Chinese typical culture collection center on March 1st, 2009, and its deposit number is CTCC NO:M 209035.
The physiological and biochemical property of above-mentioned Saccharomyces cerevisiae P-3CTCC NO:M 209035 bacterial strains is as shown in table 1.
The physiological and biochemical property of table 1 bacterial strain Saccharomyces cerevisiae P-3CTCC NO:M 209035
The 18S rDNA sequence of above-mentioned Saccharomyces cerevisiae P-3CTCC NO:M 209035 bacterial strains and the 18S rDNA sequence of other many Accharomyces cerevisiaes have 99% similarity.
Above-mentioned Saccharomyces cerevisiae P-3CTCC NO:M 209035 bacterial strains; The method that is used for carrying out bioconversion on L-phenylalanine to produce 2-phenethyl alcohol: with MPK substratum carrying out bioconversion on L-phenylalanine to produce 2-phenethyl alcohol; Used MPK culture medium prescription is: molasses 20～50 grams per liters; L-phenylalanine(Phe) 5～12 grams per liters, potassium primary phosphate 0.5 grams per liter is regulated pH to 7.0.Sterilized 20 minutes for 115 ℃.Invert point is 35 ℃, in 500 milliliters Erlenmeyer flask, adorns 75 milliliters of MPK substratum, places on the shaking table with 180 rev/mins rotating speed to cultivate 40 hours, can obtain sophisticated 2 phenylethyl alcohol conversion fluid.
Saccharomyces cerevisiae P-3CTCC NO:M 209035 bacterial strains provided by the invention can be used for microbe transformation method and produce 2 phenylethyl alcohol.Microbe transformation method production 2 phenylethyl alcohol has the reaction conditions gentleness, Product Green is natural, cost is lower, advantages of environment protection.
Embodiment
Embodiment one:
The screening of step 1:Saccharomyces cerevisiae P-3CTCC NO:M 209035 bacterial strains
Get garden and amino acid factory soil on every side; Take by weighing 5 grams and be dissolved in 100 ml physiological salines; Fully get 10 milliliters behind the mixing and add in 50 milliliters of MPK substratum, under 35 ℃ of conditions, 180 rev/mins of shaking culture are after 48 hours; The fresh MPK culture medium culturing of inoculum size with 6% (volume) switching 24 hours repeats this process 3～5 times.The bacterium liquid dilution of cultivating is coated on the nutrient agar for 1,000,000 times, under 35 ℃ of conditions, left standstill and cultivated 20 hours.The nutrient agar prescription is: peptone 10 grams per liters, Carnis Bovis seu Bubali cream 3 grams per liters, sodium-chlor 5 grams per liters, agar 18 grams per liters.The original ph of substratum is 7.2,115 ℃ of sterilizations 20 minutes.Choose in the MPK substratum of 50 milliliters of single colony inoculations of growing; Shaking culture screening in 24 hours can transform the bacterial strain of L-phenylalanine(Phe) generation 2 phenylethyl alcohol and measure 2 phenylethyl alcohol content under 35 ℃ of conditions; The final bacterial strain that a strain conversion capability is better, can accumulate the higher concentration 2 phenylethyl alcohol, i.e. the Saccharomyces cerevisiaeP-3 of obtaining.
Show by the Physiology and biochemistry identification experiment; The strain morphology that obtains through above-mentioned experiment is ellipse or oval, and 5～10 microns of diameters, colony colour are light yellow; Glucose capable of using, semi-lactosi, SANMALT-S, sucrose, seminose; Can not utilize lactose and cellobiose, can reduce fat and gelatin, can not utilize Citrate trianion, nitrate salt, the blue B of diazo and urea; Can be to grow on 10% the NaCl substratum with containing mass percent not containing VITAMINs, the 18S rDNA sequence of its 18S rDNA sequence and other many Accharomyces cerevisiaes has 99% similarity.
This bacterial strain called after Saccharomyces cerevisiae P-3 is preserved in Chinese typical culture collection center on March 1st, 2009, and its deposit number is CTCC NO:M 209035.
Step 2: the cell liquid culture for preparing above-mentioned Saccharomyces cerevisiae P-3CTCC NO:M 209035 bacterial strains
Picking is with Saccharomyces cerevisiae P-3CTCC NO:M209035 bacterial strain one ring of nutrient agar slant culture; Be seeded in 500 milliliters of Erlenmeyer flasks that sterilized 50 milliliters of nutrient broth mediums are housed; Cultivated 24 hours on shaking table in 35 ℃, 180 rev/mins, promptly make the cell liquid culture of Saccharomyces cerevisiae P-3CTCC NO:M 209035 bacterial strains.
Above-mentioned nutrient broth medium is identical with the preparation method of nutrient agar, does not just add agar.
Step 3: utilize above-mentioned Saccharomyces cerevisiae P-3CTCC NO:M 209035 bacterial strains, obtain sophisticated 2 phenylethyl alcohol conversion fluid to cultivate in the MPK substratum that contains 20 grams per liter molasses
The cell liquid culture of the Saccharomyces cerevisiae P-3CTCC NO:M209035 bacterial strain that will obtain through the method for above-mentioned steps 1,2; With volume percent is that 6% inoculum size is inoculated in 500 milliliters of Erlenmeyer flasks that sterilized 75 milliliters of MPK substratum are housed, and cultivates on shaking table in 35 ℃, 180 rev/mins.Concentration at the 40th hour 2 phenylethyl alcohol reaches 2.7 grams per liters, promptly gets sophisticated 2 phenylethyl alcohol conversion fluid.
The above-mentioned MPK culture medium prescription that contains 20 grams per liter molasses is: molasses 20 grams per liters, and L-phenylalanine(Phe) 7 grams per liters, potassium primary phosphate 0.5 grams per liter is regulated pH to 7.0.Sterilized 20 minutes for 115 ℃.
Embodiment two: utilize above-mentioned Saccharomyces cerevisiae P-3CTCC NO:M 209035 bacterial strains, obtain sophisticated 2 phenylethyl alcohol conversion fluid to cultivate in the MPK substratum that contains 30 grams per liter molasses
Identical with the step of the foregoing description one, in the prescription of different just MPK substratum molasses concentration be 30 grams per liters.Obtained sophisticated conversion fluid at the 40th hour, wherein the concentration of 2 phenylethyl alcohol reaches 3.4 grams per liters.
Embodiment three: utilize above-mentioned Saccharomyces cerevisiae P-3CTCC NO:M 209035 bacterial strains, obtain sophisticated 2 phenylethyl alcohol conversion fluid to cultivate in the MPK substratum that contains 40 grams per liter molasses
Identical with the step of the foregoing description one, in the prescription of different just MPK substratum molasses concentration be 40 grams per liters.Obtained sophisticated conversion fluid at the 40th hour, wherein the concentration of 2 phenylethyl alcohol reaches 3.8 grams per liters.
Embodiment four: utilize above-mentioned Saccharomyces cerevisiae P-3CTCC NO:M 209035 bacterial strains, obtain sophisticated 2 phenylethyl alcohol conversion fluid to cultivate in the MPK substratum that contains 50 grams per liter molasses
Identical with the step of the foregoing description one, in the prescription of different just MPK substratum molasses concentration be 50 grams per liters.Obtained sophisticated conversion fluid at the 40th hour, wherein the concentration of 2 phenylethyl alcohol reaches 4.1 grams per liters.
Embodiment five: utilize above-mentioned Saccharomyces cerevisiae P-3CTCC NO:M 209035 bacterial strains, obtain sophisticated 2 phenylethyl alcohol conversion fluid to cultivate in the MPK substratum that contains 30 grams per liter molasses and 5 grams per liter L-phenylalanine(Phe)s
Identical with the step of the foregoing description one, in the prescription of different just MPK substratum the L-phenyl-alanine concentration be 5 grams per liters, molasses concentration is 30 grams per liters.Obtained sophisticated conversion fluid at the 40th hour, wherein the concentration of 2 phenylethyl alcohol reaches 3.3 grams per liters.
Embodiment six: utilize above-mentioned Saccharomyces cerevisiae P-3CTCC NO:M 209035 bacterial strains, obtain sophisticated 2 phenylethyl alcohol conversion fluid to cultivate in the MPK substratum that contains 30 grams per liter molasses and 12 grams per liter L-phenylalanine(Phe)s
Identical with the step of the foregoing description one, in the prescription of different just MPK substratum the L-phenyl-alanine concentration be 12 grams per liters, molasses concentration is 30 grams per liters.Obtained sophisticated conversion fluid at the 40th hour, wherein the concentration of 2 phenylethyl alcohol reaches 3.6 grams per liters.
Claims (3)
1. an Accharomyces cerevisiae; It is characterized in that: the bacterial strain of this yeast saccharomyces cerevisiae is called Saccharomyces cerevisiae P-3; Be preserved in Chinese typical culture collection center on March 1st, 2009, its deposit number is CTCC NO:M209035, and the characteristic of these Saccharomyces cerevisiae P-3CTCC NO:M 209035 bacterial strains is: be shaped as ellipse or oval; 5～10 microns of diameters; Colony colour is light yellow, and glucose capable of using, semi-lactosi, SANMALT-S, sucrose, seminose can reduce fat and gelatin; Is to grow on 10% the NaCl substratum not containing VITAMINs with containing mass percent, and the 18S rDNA sequence of said Saccharomyces cerevisiae P-3CTCC NO:M 209035 bacterial strains and the 18S rDNA sequence of other many Accharomyces cerevisiaes have 99% similarity.
2. yeast saccharomyces cerevisiae as claimed in claim 1 is used for the method for carrying out bioconversion on L-phenylalanine to produce 2-phenethyl alcohol; It is characterized in that: said Saccharomyces cerevisiae P-3CTCC NO:M 209035 bacterial strains are with MPK substratum carrying out bioconversion on L-phenylalanine to produce 2-phenethyl alcohol; Said MPK culture medium prescription is: molasses 20～50 grams per liters; L-phenylalanine(Phe) 5～12 grams per liters, potassium primary phosphate 0.5 grams per liter.
3. yeast saccharomyces cerevisiae as claimed in claim 2 is used for the method for carrying out bioconversion on L-phenylalanine to produce 2-phenethyl alcohol; It is characterized in that: with said Saccharomyces cerevisiae P-3CTCC NO:M 209035 bacterial strains with MPK substratum carrying out bioconversion on L-phenylalanine to produce 2-phenethyl alcohol; Invert point is 35 ℃; In 500 milliliters Erlenmeyer flask, adorn 75 milliliters of MPK substratum, cultivated 40 hours, can obtain sophisticated 2 phenylethyl alcohol conversion fluid with 180 rev/mins rotating speeds.
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| TWI735195B (en) * | 2020-03-30 | 2021-08-01 | 財團法人食品工業發展研究所 | Saccharomyces cerevisiaeand method for producing 2-phenylethanol or beer by using the same |
| CN112029672A (en) * | 2020-09-09 | 2020-12-04 | 云南瑞升烟草技术（集团）有限公司 | Method for preparing tobacco flavor by using saccharomyces cerevisiae and application of tobacco flavor in cigarette leaf group |
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| CN101363030A (en) * | 2008-09-18 | 2009-02-11 | 福州大学 | Method for preparing 2-phenethyl alcohol coupling yeast transformation and resin adsorption |
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| CN101363030A (en) * | 2008-09-18 | 2009-02-11 | 福州大学 | Method for preparing 2-phenethyl alcohol coupling yeast transformation and resin adsorption |
Non-Patent Citations (3)
| Title |
|---|
| 崔志峰 等.酵母生物转化合成2-苯乙醇的培养条件优化.《食品与发酵工业》.2008,第34卷(第8期),52-55. * |
| 陆军 等.两相体系生物转化L-苯丙氨酸生成2-苯乙醇.《化工进展》.2008,第27卷(第3期),417-420. * |
| 黄亚东 等.关于啤酒酵母产生苯乙醇的研究.《酿酒科技》.2003,(第5期),62-63. * |
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