CN109971797A - Utilize the method for rhodotorula glutinis production tyrosol - Google Patents
Utilize the method for rhodotorula glutinis production tyrosol Download PDFInfo
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- CN109971797A CN109971797A CN201711452721.6A CN201711452721A CN109971797A CN 109971797 A CN109971797 A CN 109971797A CN 201711452721 A CN201711452721 A CN 201711452721A CN 109971797 A CN109971797 A CN 109971797A
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Abstract
The invention belongs to tyrosol production technical fields, provide a kind of method with rhodotorula glutinis production tyrosol, and rhodotorula glutinis (Rhodotorulaglutinis) is seeded in culture medium according to inoculum concentration for 4%-10%；Shaker fermentation is carried out under the conditions of 28-32 DEG C, revolving speed is 100~180r/min, cultivates 12~16h；Then the L-phenylalanine solution that addition mass concentration is 2% adds 5% that liquor capacity is culture volume every time, and addition number is 2~4 times, and for the adjacent time interval added twice in 20~30h, temperature is down to 20~25 DEG C of fermentations, and ferment 40~120h；Fermentation liquid is subjected to vacuum freeze-drying processing after fermentation, 6~12h of methanol solution extraction is added into freeze dried substance；Extract liquor centrifugation removal of impurities, supernatant concentration；Concentrate is detected through silica gel column chromatography and thin-layer chromatography, is collected the eluent that principal spot is tyrosol and is recrystallized to obtain tyrosol product.The present invention utilizes sticky red rhodotorula biosynthesis tyrosol, and the period is short, it can be achieved that continuous production, production method is simple, efficiently.
Description
Technical field
It is the present invention relates to a kind of microbial production of tyrosol, in particular to a kind of to produce tyrosol using rhodotorula glutinis
Method.
Background technique
Tyrosol (p-hydroxyphenylethanol, p-tyrosol), belongs to typical phenol natural, is medicinal red scape
One of effective component of its plant itself has and prevents cardiovascular, antitumor, anti-inflammatory and antiallergic isoreactivity, can also be used as
Precursor compound is used to prepare the drug with curative effects such as treatment hypertension, heart failure and anti-cancers.As people make its pharmacology
Understanding deepens continuously, and market demand is growing, and whether consumer belongs to biological source more focused on it.
Currently, the production method of tyrosol relies primarily on rhodiola plant extraction or chemical synthesis, such as Jiangsu Zhong Kang drug
" tyrosol and tyrosol by-product salidroside plant extraction and its preparation and purposes " (patent application disclosed in Science and Technology Ltd.
Number: 200610096054.8), disclosed in Shenyang Pharmaceutical University " to be raw material through benzyl protection, hydrocarbonylation, debenzylation system to bromine phenol
Tyrosol " method (Chinese journal of Medicinal Chemistry, 2002) and 2012 " Guangdong chemical industry " report " with phenol and its derivatives
It is Material synthesis tyrosol with benzyl carbinol " etc..In recent years, the method that people begin to focus on microbial method production tyrosol, such as Chinese section
Tianjin Institute of Industrial Biotechnology, institute discloses " a kind of method and application of the biosynthesis tyrosol in Escherichia coli " (specially
Sharp application number: 201310133238.7) it, and using the method for rhodotorula glutinis synthesis tyrosol is not reported so far.Rhodotorula glutinis because
Closely related phenylalanine metabolic pathways approach is synthesized with tyrosol, and its phenylalanine lyase PAL activity is very high, be synthesis
The potentiality bacterial strain of tyrosol.
Summary of the invention
It is an object of that present invention to provide the methods of rhodotorula glutinis production tyrosol.It is the bacterium for producing tyrosol with sticky red rhodotorula
Kind, tyrosol is obtained by fermentation control and separation and Extraction.
A kind of method with sticky red rhodotorula production tyrosol provided according to the present invention, it is described to produce junket with sticky red rhodotorula
The method of alcohol specifically comprises the following steps:
(1), rhodotorula glutinis (Rhodotorula glutinis) is seeded to culture medium according to inoculum concentration for 4~10%
In, every 1000ml culture medium includes the following raw material: 20~60g of glucose, 1~2g of yeast powder, 5~8g of potassium dihydrogen phosphate, sulfuric acid
1~3g of ammonium, 1~3g of sodium sulphate, 0.2~0.5g of magnesium sulfate, surplus are water；
It (2), is 28~32 DEG C in temperature after inoculation, revolving speed is that 12~16h is cultivated under 100~180r/min；
(3), L-phenylalanine is added in the culture solution of Xiang Suoshu step (2), additive amount is that the step (1) culture medium is every
2~5g is added in 1000ml, adds 40~120h of post-fermentation；
(4), fermentation liquid is subjected to vacuum freeze-drying processing after fermentation, is added into the substance after freeze-drying and is equivalent to fermentation
The methanol solution of liquid product 10~30% is 15~30 DEG C in temperature, and revolving speed is that 6~12h is extracted under 30~60r/min；
(5), by the extract liquor of the step (4) temperature be 2~8 DEG C, revolving speed be 4000~8000r/min under carry out from
Heart removal of impurities, stays supernatant to be concentrated, obtains head product.
(6), by the head product by silica gel chromatography gradient elution of the step (5), thin-layer chromatography detection collects principal spot and is
The eluent of tyrosol recrystallizes to obtain white plates tyrosol product.
Preferably, it is 2% solution that the L-phenylalanine of step (3) addition, which is mass concentration, and addition number is 2~5
Secondary, the time interval added every time is in 20~30h.
Preferably, after the addition L-phenylalanine solution, fermentation temperature is down to 20~25 DEG C.
Specific embodiment
Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate the present invention
Rather than it limits the scope of the invention.
Embodiment 1
A kind of method with sticky red rhodotorula production tyrosol provided in this embodiment, it is described to produce tyrosol with sticky red rhodotorula
Method specifically comprise the following steps:
(1), rhodotorula glutinis (Rhodotorulaglutinis) is seeded in culture medium according to inoculum concentration for 4%, often
1000ml culture medium includes the following raw material: glucose 20g, yeast powder 1g, potassium dihydrogen phosphate 5g, ammonium sulfate 1g, sodium sulphate 1g, sulphur
Sour magnesium 0.2g, surplus are water；
It (2), is 28 DEG C in temperature after inoculation, revolving speed is to cultivate 16h under 100r/min；
(3), L-phenylalanine 2g is added in the culture solution of Xiang Suoshu step (2), adds post-fermentation 40h；
(4), fermentation liquid is subjected to vacuum freeze-drying processing after fermentation, is added into the substance after freeze-drying and is equivalent to fermentation
The methanol solution of liquid product 10% is 15 DEG C in temperature, and revolving speed is to extract 6h under 60r/min；
It (5), is 2 DEG C in temperature by the extract liquor of the step (4), revolving speed is to carry out centrifugation removal of impurities under 4000r/min, is stayed
Supernatant concentration, obtains head product.
(6), by the head product by silica gel chromatography gradient elution of the step (5), thin-layer chromatography detection collects principal spot and is
The eluent of tyrosol recrystallizes to obtain white plates tyrosol product.
The L-phenylalanine of step (3) addition is that mass concentration is 2% solution, and addition number is 2 times, is added every time
Time interval in 20~30h.
After the addition L-phenylalanine solution, fermentation temperature is down to 20 DEG C.
Embodiment 2
A kind of method with sticky red rhodotorula production tyrosol provided in this embodiment, it is described to produce tyrosol with sticky red rhodotorula
Method specifically comprise the following steps:
(1), rhodotorula glutinis (Rhodotorulaglutinis) is seeded in culture medium according to inoculum concentration for 10%, often
1000ml culture medium includes the following raw material: glucose 60g, yeast powder 2g, potassium dihydrogen phosphate 8g, ammonium sulfate 3g, sodium sulphate 3g, sulphur
Sour magnesium 0.5g, surplus are water；
It (2), is 32 DEG C in temperature after inoculation, revolving speed is to cultivate 16h under 180r/min；
(3), L-phenylalanine is added in the culture solution of Xiang Suoshu step (2), additive amount is that the step (1) culture medium is every
5g is added in 1000ml, adds post-fermentation 120h；
(4), fermentation liquid is subjected to vacuum freeze-drying processing after fermentation, is added into the substance after freeze-drying and is equivalent to fermentation
The methanol solution of liquid product 20% is 30 DEG C in temperature, and revolving speed is to extract 12h under 30r/min；
It (5), is 8 DEG C in temperature by the extract liquor of the step (4), revolving speed is to carry out centrifugation removal of impurities under 8000r/min, is stayed
Supernatant concentration, obtains head product.
(6), by the head product by silica gel chromatography gradient elution of the step (5), thin-layer chromatography detection collects principal spot and is
The eluent of tyrosol recrystallizes to obtain white plates tyrosol product.
The L-phenylalanine of step (3) addition is that mass concentration is 2% solution, and addition number is 5 times, is added every time
Time interval for 24 hours.
After the addition L-phenylalanine solution, fermentation temperature is down to 25 DEG C.
Embodiment 3
A kind of method with sticky red rhodotorula production tyrosol provided in this embodiment, it is described to produce tyrosol with sticky red rhodotorula
Method specifically comprise the following steps:
(1), rhodotorula glutinis (Rhodotorulaglutinis) is seeded in culture medium according to inoculum concentration for 6%, often
1000ml culture medium includes the following raw material: glucose 40g, yeast powder 1.5g, potassium dihydrogen phosphate 6g, ammonium sulfate 2g, sodium sulphate 2g,
Magnesium sulfate 0.3g, surplus are water；
It (2), is 30 DEG C in temperature after inoculation, revolving speed is to cultivate 14h under 140r/min；
(3), L-phenylalanine is added in the culture solution of Xiang Suoshu step (2), additive amount is that the step (1) culture medium is every
3g is added in 1000ml, adds post-fermentation 90h；
(4), fermentation liquid is subjected to vacuum freeze-drying processing after fermentation, is added into the substance after freeze-drying and is equivalent to fermentation
The methanol solution of liquid product 30% is 25 DEG C in temperature, and revolving speed is to extract 10h under 40r/min；
It (5), is 4 DEG C in temperature by the extract liquor of the step (4), revolving speed is to carry out centrifugation removal of impurities under 5000r/min, is stayed
Supernatant concentration, obtains head product.
(6), by the head product by silica gel chromatography gradient elution of the step (5), thin-layer chromatography detection collects principal spot and is
The eluent of tyrosol recrystallizes to obtain white plates tyrosol product.
The L-phenylalanine of step (3) addition is that mass concentration is 2% solution, and addition number is 3 times, is added every time
Time interval in 30h.
After the addition L-phenylalanine solution, fermentation temperature is down to 24 DEG C.
Claims (3)
1. a kind of method with rhodotorula glutinis production tyrosol, which is characterized in that described method includes following steps:
(1), rhodotorula glutinis (Rhodotorula glutinis) is seeded in culture medium according to inoculum concentration for 4~10%, often
1000ml culture medium includes the following raw material: 20~60g of glucose, 1~2g of yeast powder, 5~8g of potassium dihydrogen phosphate, ammonium sulfate 1~
3g, 1~3g of sodium sulphate, 0.2~0.5g of magnesium sulfate, surplus are water, and the pH value of the culture medium is between 5~7；
It (2), is 28~32 DEG C in temperature after inoculation, revolving speed is that 12~16h is cultivated under 100~180r/min；
(3), L-phenylalanine solution is added in the culture solution of Xiang Suoshu step (2), additive amount is the step (1) culture substrate
Measuring score is 2~8%, adds 40~96h of post-fermentation；
(4), fermentation liquid is subjected to vacuum freeze-drying processing after fermentation, is added into the substance after freeze-drying and is equivalent to fermented liquid
The methanol solution of product 10~30% is 15~30 DEG C in temperature, and revolving speed is that 6~12h is extracted under 30~60r/min；
It (5), is 2~8 DEG C in temperature by the extract liquor of the step (4), revolving speed is to carry out centrifugation under 4000~8000r/min to remove
It is miscellaneous, it stays supernatant to be concentrated, obtains head product.
(6), by the head product by silica gel chromatography gradient elution of the step (5), it is tyrosol that principal spot is collected in thin-layer chromatography detection
Drop saliva recrystallize to obtain white plates tyrosol product.
2. the method according to claim 1 with rhodotorula glutinis production tyrosol, which is characterized in that step (3) addition
Its mass concentration of L-phenylalanine solution be 1~3%, addition number is 2~4 times, the time interval added every time 20~
30h。
3. the method according to claim 2 with rhodotorula glutinis production tyrosol, which is characterized in that addition L-phenylalanine is molten
After liquid, fermentation temperature is down to 20~25 DEG C.
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| CN201711452721.6A CN109971797B (en) | 2017-12-28 | 2017-12-28 | Method for producing tyrosol by using rhodotorula glutinis |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110982716A (en) * | 2019-12-17 | 2020-04-10 | 厦门欧米克生物科技有限公司 | Strain for producing natural tyrosol and preparation method of natural tyrosol |
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| 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 |
| JP2011206006A (en) * | 2010-03-30 | 2011-10-20 | Yamagata Prefecture | Method for producing sake of new taste using mlf fermentation in combination |
| CN104099379A (en) * | 2013-04-08 | 2014-10-15 | 中国科学院天津工业生物技术研究所 | Method for biosynthesis of tyrosol in Escherichia coli and application of tyrosol |
| CN104388497A (en) * | 2014-12-01 | 2015-03-04 | 山西大学 | Method for producing salidroside and tyrosol employing phialocephala fortinii |
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| 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 |
| JP2011206006A (en) * | 2010-03-30 | 2011-10-20 | Yamagata Prefecture | Method for producing sake of new taste using mlf fermentation in combination |
| CN104099379A (en) * | 2013-04-08 | 2014-10-15 | 中国科学院天津工业生物技术研究所 | Method for biosynthesis of tyrosol in Escherichia coli and application of tyrosol |
| CN104388497A (en) * | 2014-12-01 | 2015-03-04 | 山西大学 | Method for producing salidroside and tyrosol employing phialocephala fortinii |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110982716A (en) * | 2019-12-17 | 2020-04-10 | 厦门欧米克生物科技有限公司 | Strain for producing natural tyrosol and preparation method of natural tyrosol |
| CN110982716B (en) * | 2019-12-17 | 2021-11-02 | 厦门欧米克生物科技有限公司 | Strain for producing natural tyrosol and preparation method of natural tyrosol |
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