CN110982716B - Strain for producing natural tyrosol and preparation method of natural tyrosol - Google Patents
Strain for producing natural tyrosol and preparation method of natural tyrosol Download PDFInfo
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- CN110982716B CN110982716B CN201911300530.7A CN201911300530A CN110982716B CN 110982716 B CN110982716 B CN 110982716B CN 201911300530 A CN201911300530 A CN 201911300530A CN 110982716 B CN110982716 B CN 110982716B
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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
- C12N1/14—Fungi; Culture media therefor
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- C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
- C12P7/22—Preparation of oxygen-containing organic compounds containing a hydroxy group aromatic
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Abstract
The invention discloses a strain for producing natural tyrosol and a preparation method of the natural tyrosol, wherein the strain is saccharomycete sp. The preparation method of natural tyrosol is to use the strain to produce by a microbial fermentation method. The fermentation method comprises the steps of strain activation, seed culture, fermentation, extraction and the like. The method has the advantages of low temperature and low pressure, safety, simple operation and less pollution, and is a safe and environment-friendly production method.
Description
Technical Field
The invention relates to a strain capable of producing synthetic natural tyrosol and a preparation method of the natural tyrosol.
Background
Tyrosol (p-hydroxyphenylethyl alcohol) is widely found in olive oil, soy sauce, raspberry and fermented products such as beer, fruit wine, sake. Is an important intermediate of medicine and perfume, and is widely used for synthesizing surface modifier, information storage material, food additive and the like.
The current methods for producing tyrosol mainly comprise a chemical synthesis method and a plant extraction method. The chemical synthesis method has the defects of large environmental pollution, harsh process conditions, high equipment requirement, low yield and the like. The content of the plant is low, the extraction is complex, and the supply amount of the tyrosol is limited. The productivity can not meet the market demand.
Ehrlich F proposed in 1911 the biosynthesis of tyrosol using a biosynthetic process. Elsden S R et al, 1958, proposed that tyrosol synthesis was produced by yeast, bacteria and some fungi during fermentation at yields around 200 mg/L. On the other hand, the yield of the Candida fermentation by Mohammed A S and the like is about 50 percent relative to tyrosine, but the yield does not reach the level of g/L, only stays at the level of mg/L, and does not meet the requirement of industrialization.
People increasingly advocate natural products and exclude the use of chemically synthesized chemicals, so that the research on a production method for synthesizing tyrosol by a biological method is imperative.
Disclosure of Invention
One of the purposes of the invention is to provide a yeast, Saecharomyces sp.OMK-58, which has the capability of synthesizing natural tyrosol. The preservation date is 7 months and 13 days in 2018, the preservation place is Wuhan city, Hubei province in China, Wuhan university, the preservation organization is China Center for Type Culture Collection (CCTCC), the preservation number is CCTCC NO: m2018479.
Still another object of the present invention is to provide the use of the yeast Saecharomyces sp.omk-58 for the production of natural tyrosol.
The invention also aims to provide a production method of natural tyrosol, which utilizes the saccharomyces sp.
Wherein the production method is a microbial fermentation method.
Preferably, the fermentation substrate of the microbial fermentation process is tyrosine.
Preferably, the method for producing natural tyrosol comprises the following steps:
1) the yeast species Saecharomyces sp.
2) Seed culture;
3) fermentation culture: inoculating the seed liquid cultured to logarithmic phase into a fermentation culture medium in a volume ratio of 5-20% under aseptic conditions, wherein the fermentation culture medium comprises the following components in percentage by mass: 2.0-5.0 parts of sugar raw material, 0.2-0.5 part of ammonium sulfate, 0.05-0.1 part of magnesium sulfate, 0.2-0.5 part of monopotassium phosphate, 0.1-1.5 parts of yeast extract powder and 0.5-2.0 parts of tyrosine, and fermenting to obtain tyrosol-containing fermentation liquor.
Preferably, the initial pH value of the fermentation medium is 5.0-8.0, and the fermentation is carried out for 48-72h at the temperature of 30-37 ℃, the stirring speed of 200-.
Preferably, the sugar material is at least one of glucose, sucrose and starch.
Preferably, the tyrosine in step 3) is added to the culture medium at one time or fed in batches.
Preferably, step 3) is followed by step 4) of separating tyrosol from the fermentation broth.
Compared with the background technology, the technical scheme has the following advantages:
the yeast provided by the invention can be used for producing natural tyrosol by a fermentation method, tyrosine is used as a substrate during fermentation, a target product is generated by microbial metabolism, and the method is low in temperature and pressure, relatively safe, simple to operate, less in pollution and safe and environment-friendly.
Drawings
The invention is further illustrated by the following figures and examples.
FIG. 1 is a chromatogram of a tyrosol standard.
FIG. 2 is a chromatogram of a tyrosine standard.
FIG. 3 is a chromatogram of the fermentation broth of example 2.
Detailed Description
1. Bacterial strain
The experiment takes baker's yeast as an initial strain, and a mutant strain is directionally screened out through chemical mutagenesis, and can convert L-tyrosine to generate tyrosol. The strain is preserved in China center for type culture Collection in 2018, 7 months and 13 days, and the preservation number is CCTCC NO: m2018479.
2. Strain activation
Drawing a full ring of bacteria liquid from a glycerol tube under aseptic condition, uniformly coating the bacteria liquid on a solid slant culture medium, and culturing the bacteria liquid in a biochemical incubator at 25-30 ℃ for 24-48 h. The solid slant culture medium is a wort culture medium.
3. Seed culture
Under the aseptic condition, a full ring of strains with good growth vigor is drawn on a solid culture medium by an inoculating needle and is inoculated into an aseptic seed culture medium, the initial pH value of the seed culture medium is 5.0-8.0, and the thalli are cultured to the logarithmic growth phase under the conditions of the culture temperature of 28-35 ℃ and the rotation speed of 150-. The seed culture medium comprises the following components in percentage by mass: (%)
The sugar material may be one of glucose, sucrose, starch, etc. 1.0-3.5, potassium dihydrogen phosphate 0.2-1.0, dipotassium hydrogen phosphate 0.1-0.5, sodium chloride 0.2-0.8, yeast extract powder 0.1-1.0, and peptone 0.1-1.0;
4. fermentation of
The seed solution cultured to logarithmic growth phase is inoculated into a fermentation medium under aseptic conditions at a volume ratio of 10%. The initial pH value of the fermentation medium is 5.0-8.0, preferably 5.0-6.0, and the fermentation is carried out for 48-72h under the conditions that the temperature is 30-37 ℃, the stirring speed is 200 and 500rpm, and the ventilation volume is 1: 0.05-0.15;
the fermentation formula comprises the following components in percentage by mass: (%)
The sugar material may be glucose, sucrose, starch 2.0-5.0, ammonium sulfate 0.2-0.5, magnesium sulfate 0.05-0.1, potassium dihydrogen phosphate 0.2-0.5, yeast extract powder 0.1-1.5, and tyrosine 0.5-2.0.
Tyrosine can be added into the culture medium at one time or fed batch.
5. Detection of
The tyrosol High Performance Liquid Chromatography (HPLC) detection method comprises the following steps: a chromatographic column: COSMOSIL 5C18-PAQ (4.6ID 250 mm); mobile phase: acetonitrile: 0.15% aqueous phosphoric acid 70:30 (V/V); the flow rate is 0.8 mL/min; the detection wavelength is 216 nm; the column temperature is 35 ℃; the sample volume was 5. mu.L, and the maps are shown in FIGS. 1 to 3.
Example 1: production of natural tyrosol (triangular flask oscillation fermentation)
Preparing a seed culture medium, wherein the composition of the culture medium is as follows (g/L): 10 portions of glucose, 2.0 portions of monopotassium phosphate, 1.0 portion of dipotassium phosphate, 2.5 portions of sodium chloride, 10.0 portions of yeast extract powder, 5.0 portions of peptone and water as solvent, and the initial pH value is 6.0. Sterilizing at 121 deg.C for 15 min.
Preparing a fermentation medium, wherein the composition of the medium is as follows (g/L): 30 parts of glucose, 4 parts of monopotassium phosphate, 3.0 parts of ammonium sulfate, 0.8 part of magnesium sulfate, 1.0 part of yeast extract powder, 10.0 parts of tyrosine, water as a solvent and 7.0 parts of initial pH value. Sterilizing at 121 deg.C for 15 min.
Preparing seeds: aseptically transferring the low-temperature glycerinum tube OMK-58 strain onto a fresh and aseptic solid plate, culturing for 24h at 30 ℃, selecting a bacterial colony, inoculating into a 500mL triangular flask for seed culture, wherein the volume of a seed culture medium in the flask is 50mL, the rotating speed of a shaking table is 180rpm, and culturing for 16h at 30 ℃ to obtain a seed solution.
Fermentation of natural tyrosol: 50mL of prepared fermentation medium is poured into a 500mL sterile triangular flask, then 7.5mL of cultured seed solution is inoculated for fermentation, the fermentation temperature is 30 ℃, and the rotation speed of a shaking table is 200 rpm. After fermenting for 100h, the concentration of the natural tyrosol in the fermentation liquor is measured to be 2.0g/L by an HPLC method.
Example 2: production of Natural tyrosol (20L bioreactor fermentation)
The seed culture medium and the fermentation medium were prepared in the same manner as in example 1.
Preparing seeds: aseptically transferring the strain OMK-58 of the low-temperature glycerin tube on a fresh and aseptic solid plate, culturing for 24h at 30 ℃, selecting a colony, inoculating the colony in a 3L seeding tank filled with 1.8L seed culture medium for seed culture, ventilating and stirring at the rotating speed of 300rpm, and culturing for 18h at 30 ℃ to obtain a seed solution.
Fermentation of natural tyrosol: and (2) putting 10.2L of prepared fermentation medium into a 20L fermentation tank, sterilizing at 121 ℃ for 15min, inoculating 1.8L of cultured seed liquid into the 20L fermentation tank for fermentation, wherein the fermentation temperature is 30 ℃, the stirring speed is 400rpm, and the aeration ratio is 1: fermenting for 0.1 to 60 h. After the fermentation, the concentration of the natural tyrosol in the fermentation broth is measured to be 5.0g/L by an HPLC method.
Claims (8)
1. Yeast (Saecharomyces sp. omk-58), characterized by: the preservation number of the strain in China center for type culture Collection is CCTCC NO: m2018479.
2. Use of the yeast (Saecharomyces sp. omk-58) according to claim 1 for the production of natural tyrosol.
3. A production method of natural tyrosol is characterized in that: production is carried out using the yeast of claim 1 (Saecharomyces sp. omk-58) and tyrosine is the substrate of the fermentation.
4. A process for the production of natural tyrosol according to claim 3 comprising the steps of:
1) activating yeast (Saccharomyces sp. OMK-58) strain;
2) seed culture;
3) fermentation culture: inoculating the seed liquid cultured to logarithmic phase into a fermentation culture medium in a volume ratio of 5-20% under aseptic conditions, wherein the fermentation culture medium comprises the following components in percentage by mass: 2.0-5.0 parts of sugar raw material, 0.2-0.5 part of ammonium sulfate, 0.05-0.1 part of magnesium sulfate, 0.2-0.5 part of monopotassium phosphate, 0.1-1.5 parts of yeast extract powder and 0.5-2.0 parts of tyrosine, and fermenting to obtain tyrosol-containing fermentation liquor.
5. A process according to claim 4, wherein the natural tyrosol is produced by: step 2) the seed culture medium used for seed culture comprises the following components in percentage by mass: 1.0-3.5 parts of sugar raw material, 0.2-1.0 part of potassium dihydrogen phosphate, 0.1-0.5 part of dipotassium hydrogen phosphate, 0.2-0.8 part of sodium chloride, 0.1-1.0 part of yeast extract powder and 0.1-1.0 part of peptone.
6. A process according to claim 4 or 5, wherein the process comprises: the initial pH value of the fermentation medium is 5.0-8.0, and the fermentation is carried out for 48-72h at the temperature of 30-37 ℃, the stirring speed of 200-.
7. A process according to claim 4 or 5, wherein the process comprises: the sugar material is at least one of glucose, sucrose and starch.
8. A process according to claim 4 or 5, wherein the process comprises: and 3) tyrosine is added into the culture medium at one time or fed-batch.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2004215644A (en) * | 2002-11-20 | 2004-08-05 | Yamagata Prefecture | Thyrosol-producing mutant yeast strain having high productivity, and method for producing fermented alcoholic beverage using the yeast |
| JP2012085614A (en) * | 2010-10-22 | 2012-05-10 | Saga Univ | Mutant yeast strain having high tyrosol productivity, and method for producing fermented alcoholic beverage using the same |
| CN108753636A (en) * | 2018-06-12 | 2018-11-06 | 山东恒鲁生物科技有限公司 | A kind of yeast and construction method producing tyrosol and hydroxytyrosol |
| CN109971797A (en) * | 2017-12-28 | 2019-07-05 | 北京农学院 | Utilize the method for rhodotorula glutinis production tyrosol |
| CN110499260A (en) * | 2018-09-25 | 2019-11-26 | 成都薇合生物科技有限公司 | Engineering bacteria and its application of a kind of high yield rhodioside and/or tyrosol |
-
2019
- 2019-12-17 CN CN201911300530.7A patent/CN110982716B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2004215644A (en) * | 2002-11-20 | 2004-08-05 | Yamagata Prefecture | Thyrosol-producing mutant yeast strain having high productivity, and method for producing fermented alcoholic beverage using the yeast |
| JP2012085614A (en) * | 2010-10-22 | 2012-05-10 | Saga Univ | Mutant yeast strain having high tyrosol productivity, and method for producing fermented alcoholic beverage using the same |
| CN109971797A (en) * | 2017-12-28 | 2019-07-05 | 北京农学院 | Utilize the method for rhodotorula glutinis production tyrosol |
| CN108753636A (en) * | 2018-06-12 | 2018-11-06 | 山东恒鲁生物科技有限公司 | A kind of yeast and construction method producing tyrosol and hydroxytyrosol |
| CN110499260A (en) * | 2018-09-25 | 2019-11-26 | 成都薇合生物科技有限公司 | Engineering bacteria and its application of a kind of high yield rhodioside and/or tyrosol |
Non-Patent Citations (1)
| Title |
|---|
| 黄酒中的苦味物质及其来源探讨;杨国军;《食品与发酵工业》;20160331(第02期);37-41 * |
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