CN109439596A - The flora of one kind production L-thiamine and its application - Google Patents
The flora of one kind production L-thiamine and its application Download PDFInfo
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- CN109439596A CN109439596A CN201811545381.6A CN201811545381A CN109439596A CN 109439596 A CN109439596 A CN 109439596A CN 201811545381 A CN201811545381 A CN 201811545381A CN 109439596 A CN109439596 A CN 109439596A
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- 229960003495 thiamine Drugs 0.000 title claims abstract description 27
- 239000011721 thiamine Substances 0.000 title claims abstract description 27
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 22
- 235000009024 Ceanothus sanguineus Nutrition 0.000 claims abstract description 55
- 240000003553 Leptospermum scoparium Species 0.000 claims abstract description 55
- 235000015459 Lycium barbarum Nutrition 0.000 claims abstract description 55
- 238000000034 method Methods 0.000 claims abstract description 14
- QUSNBJAOOMFDIB-UHFFFAOYSA-N Ethylamine Chemical compound CCN QUSNBJAOOMFDIB-UHFFFAOYSA-N 0.000 claims abstract description 8
- 239000001963 growth medium Substances 0.000 claims abstract description 5
- 241000193830 Bacillus <bacterium> Species 0.000 claims abstract description 3
- 241000598860 Garcinia hanburyi Species 0.000 claims abstract description 3
- 229940117709 gamboge Drugs 0.000 claims abstract description 3
- 241001122767 Theaceae Species 0.000 claims description 10
- 238000005520 cutting process Methods 0.000 claims description 8
- 239000007788 liquid Substances 0.000 claims description 8
- 239000002609 medium Substances 0.000 claims description 8
- 239000012528 membrane Substances 0.000 claims description 8
- 238000007789 sealing Methods 0.000 claims description 8
- 239000007787 solid Substances 0.000 claims description 8
- 241000037488 Coccoloba pubescens Species 0.000 claims description 7
- 239000006137 Luria-Bertani broth Substances 0.000 claims description 7
- 238000011534 incubation Methods 0.000 claims description 6
- 239000006228 supernatant Substances 0.000 claims description 6
- 150000001412 amines Chemical class 0.000 claims description 2
- 238000005119 centrifugation Methods 0.000 claims description 2
- 238000005192 partition Methods 0.000 claims description 2
- DATAGRPVKZEWHA-YFKPBYRVSA-N N(5)-ethyl-L-glutamine Chemical compound CCNC(=O)CC[C@H]([NH3+])C([O-])=O DATAGRPVKZEWHA-YFKPBYRVSA-N 0.000 abstract description 58
- 229940026510 theanine Drugs 0.000 abstract description 28
- 239000000758 substrate Substances 0.000 abstract description 13
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 abstract description 8
- 238000003786 synthesis reaction Methods 0.000 abstract description 7
- 230000015572 biosynthetic process Effects 0.000 abstract description 6
- 230000028327 secretion Effects 0.000 abstract description 3
- 241000589636 Xanthomonas campestris Species 0.000 abstract description 2
- 230000003698 anagen phase Effects 0.000 abstract description 2
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 abstract 2
- 238000011017 operating method Methods 0.000 abstract 1
- 238000012360 testing method Methods 0.000 description 8
- 238000001514 detection method Methods 0.000 description 7
- 238000000338 in vitro Methods 0.000 description 7
- 241000894006 Bacteria Species 0.000 description 5
- 229920003266 Leaf® Polymers 0.000 description 4
- 238000004128 high performance liquid chromatography Methods 0.000 description 4
- 238000005374 membrane filtration Methods 0.000 description 4
- 244000005700 microbiome Species 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
- 238000012797 qualification Methods 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 230000003595 spectral effect Effects 0.000 description 2
- DATAGRPVKZEWHA-RXMQYKEDSA-N (2r)-2-azaniumyl-5-(ethylamino)-5-oxopentanoate Chemical compound CCNC(=O)CC[C@@H](N)C(O)=O DATAGRPVKZEWHA-RXMQYKEDSA-N 0.000 description 1
- 244000063299 Bacillus subtilis Species 0.000 description 1
- 235000014469 Bacillus subtilis Nutrition 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- DATAGRPVKZEWHA-UHFFFAOYSA-N L-gamma-glutamyl-n-ethylamine Natural products CCNC(=O)CCC(N)C(O)=O DATAGRPVKZEWHA-UHFFFAOYSA-N 0.000 description 1
- -1 N- ethyl-gamma-glutamyl amine Chemical class 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 230000036772 blood pressure Effects 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 235000019634 flavors Nutrition 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 239000002778 food additive Substances 0.000 description 1
- 235000013376 functional food Nutrition 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000001537 neural effect Effects 0.000 description 1
- 239000002574 poison Substances 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000007670 refining Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000012549 training Methods 0.000 description 1
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- 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
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- 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
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- 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
- C12N1/20—Bacteria; Culture media therefor
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- 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
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
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Abstract
It is the tea tree endophyte being separated to from tea tree sterile tissue cultivating seedling the invention discloses the flora of a kind of production L-thiamine and its application, the flora, belongs to Xanthomonas campestris section gamboge Bacillus.The flora of production L-thiamine of the invention, for the endophyte separated from tea tree for the first time, the well-grown on LB culture medium, 28 DEG C are cultivated to logarithmic growth phase, added into culture medium and continue culture 2-3 days after glutamine and ethamine, experimental group is able to detect that the synthesis of L-thiamine compared with control group, and the theanine of synthesis can rapid secretion to extracellular, secernment efficiency can efficiently synthesize secretion theanine up to 90% or more.Production L-thiamine method of the invention, using glutamine and ethamine as substrate, operating method is simple, theanine high production efficiency, has good industrial applications prospect.
Description
Technical field
The invention belongs to microorganisms technical fields, and in particular to the flora of one kind production L-thiamine and its application.
Background technique
Theanine (L-Theanine) is N- ethyl-gamma-glutamyl amine, accounts for 50% or more of total amino acid content in tealeaves,
It is distinctive nonprotein free amino acid in tea tree, is also to confer to the main matter of tealeaves flavour, content is directly determined
Determine tea leaf quality.Theanine, which has, to be loosened neural, antitumor, antidepression, protection blood vessel, blood pressure lowering, inhibits fat and improve machine
The medicine effect such as body immunity function, further, it is also possible to be applied to field of food as functional food additives.Therefore, tea ammonia
Acid is increasingly favored by people, and international market is also very big to its demand.
Currently, there are mainly three types of the methods of production theanine.One is extract from tealeaves.Theanine in Tea content compared with
It is low, and raw material sources are limited, cause that the production cost is very high.Second method is chemical synthesis.This method not only results in poison
Property substance residual, and the theanine produced is the mixture of D- theanine and L-thiamine isomers, and it is complicated to split process for refining.
The third method is to utilize microorganism and its enzyme fermentation production theanine.That reports at present, which can synthesize the microorganism of theanine, includes
Escherichia coli, bacillus subtilis, pseudomonad, but these are all nature free microorganisms, are separated out of tea tree body
It obtains.
Summary of the invention
Goal of the invention: being directed to the deficiencies in the prior art, and the object of the present invention is to provide one kind to produce L-thiamine
Flora satisfied the use demand for the endophyte that is separated from tea tree for the first time.It is a further object of the present invention to provide a kind of above-mentioned
Flora is producing the application in L-thiamine.
Technical solution: in order to achieve the above-mentioned object of the invention, the technical solution adopted by the present invention are as follows:
The flora of one kind production L-thiamine, for the tea tree endophyte being separated to from tea tree sterile tissue cultivating seedling.
The flora of the production L-thiamine, partition method are as follows: access piece tea-tree tissue culture seedling blade is cut into scissors
After the sheet of 0.5cm × 1.0cm, random oblique cutting enters in solid LB media, sealed membrane sealing, 28 DEG C of constant temperature incubation carton upside down trainings
It supports 2-3 days, after growing flora abundant around blade, is inoculated into and carries out continuing to cultivate in LB liquid medium, obtained
The flora obtained is the flora for producing L-thiamine.
The tea tree breed is Anhui Province's tea tree breed " Shu Chazao ", Yunnan Province's tea tree large leaf " cloud anti-No. 10 ", Zhejiang
River province tea tree breed " Dragon Well tea 43 " and " black ox is early ".
The flora is Xanthomonas campestris section gamboge Bacillus.
Application of the flora in production L-thiamine.
A method of L-thiamine being produced, steps are as follows:
1) the access sterile tissue culture seedling leaf of piece tea tree, after the sheet of 0.5cm × 1.0cm is cut into scissors, random oblique cutting enters
In solid LB media, sealed membrane sealing, 28 DEG C constant temperature incubation carton upside down culture 2-3 days, to grow bacterium abundant around blade
After group, it is inoculated into and carries out continuing to cultivate in LB liquid medium；
2) by flora in 28 DEG C of shaken cultivations to OD600When being 1.0, final concentration of 20mM paddy is added simultaneously into culture solution
Glutamine and 20mM ethamine cultivate 7d or more；
3) the 100 DEG C of water-baths of culture solution obtained crack 30min, and in -20 DEG C after multigelation 2~3 times, 12000rpm,
It is centrifuged 15min, obtains the supernatant for being rich in L-thiamine.
In step 2), culture 10 days or more.
The tea tree breed is Anhui Province " Shu Chazao ", Yunnan Province's tea tree large leaf " cloud anti-No. 10 ", Zhejiang Province's tealeaves
Kind " Dragon Well tea 43 " and " black ox is early ".
The utility model has the advantages that compared with prior art, the flora of production L-thiamine of the invention, to be separated from tea tree for the first time
Endophyte, the well-grown on LB culture medium, 28 DEG C of cultures add glutamine and second into culture medium to logarithmic growth phase
Continue culture 2-3 days after amine, experimental group is able to detect that the synthesis of L-thiamine compared with control group, and the theanine synthesized can be fast
Speed is secreted into extracellular, and secernment efficiency can efficiently synthesize secretion theanine up to 90% or so, has good practicability.
Detailed description of the invention
Fig. 1 is that flora figure abundant is grown around blade；
Fig. 2 is the mass spectral results figure of theanine；
Fig. 3 is the capability result figure that " Shu Chazao " tea tree endophyte flora efficiently synthesizes theanine.
Fig. 4 is that " black ox is early ", " cloud is No. 10 anti-" and " Dragon Well tea 43 " tea tree endophyte group synthesize the Mass Spectrometric Identification knot of theanine
Fruit.
Specific embodiment
The present invention is described further combined with specific embodiments below.
Embodiment 1
1) four Anhui Province Nian Sheng tea tree breed " Shu Chazao " tea tree endophyte floras are separately cultured
Superclean bench sterile working: the access sterile tissue culture seedling leaf of piece tea tree is cut into 0.5cm × 1.0cm's with scissors
After sheet, random oblique cutting enters in solid LB media, sealed membrane sealing, 28 DEG C constant temperature incubation carton upside down culture 2-3 days, to blade
Surrounding is grown after flora abundant (such as Fig. 1), is inoculated into and carries out continuing to cultivate in LB liquid medium.
2) tea tree endophyte group synthesizes the sample preparation and detection process of theanine
By flora in 28 DEG C of shaken cultivations to OD600When being 1.0, final concentration of 20mM paddy is added simultaneously into LB culture solution
Glutamine and 20mM ethamine, and respectively not add the same concentration bacterium solution of any substrate and add the blank culture with concentration of substrate
Base as a control test, under same 28 DEG C of condition of culture, continues after cultivating 1d, 3d, 5d, 7d, 10d, 15d, respectively will be different
100 DEG C of water-baths of culture solution that time point obtains crack 30min, and in -20 DEG C after multigelation 2~3 times, 12000rpm, centrifugation
15min takes supernatant after 0.22 μm of water phase membrane filtration, using high performance liquid chromatography and triple level four bars mass spectrometer into
Row Testing and appraisal.
Second order ms qualification result shows: (mass spectral results are as schemed for the stable theanine containing various concentration in culture solution
2), and at the 10th day after substrate adds, theanine content averagely about 0.36mg/L reaches as high as 0.45mg/L (such as Fig. 3).
Illustrate: the flora being separated to from four Anhui Province Nian Sheng tea tree breed " Shu Chazao " tea-tree tissue culture seedlings of in vitro culture has efficient
Synthesize the ability of theanine.
Embodiment 2
1) ten Zhejiang Province Nian Sheng tea tree breed " Dragon Well tea 43 " tea tree endophyte floras are separately cultured
Superclean bench sterile working: the access sterile tissue culture miaoye of the Zhejiang Province Pian Shiniansheng tea tree breed " Dragon Well tea 43 " tea tree
Piece, after the sheet of 0.5cm × 1.0cm is cut into scissors, random oblique cutting enters in solid LB media, sealed membrane sealing, 28 DEG C of perseverances
Temperature culture carton upside down culture 2-3 days, after growing flora abundant around blade, is inoculated into LB liquid medium and is carried out
Continue to cultivate.
2) tea tree endophyte group synthesizes the sample preparation and detection process of theanine
By flora in 28 DEG C of shaken cultivations to OD600When being 1.0, final concentration of 20mM paddy is added simultaneously into LB culture solution
Glutamine and 20mM ethamine, and respectively not add the same concentration bacterium solution of any substrate and add the blank culture with concentration of substrate
Base as a control test, under same 28 DEG C of condition of culture, continues after cultivating 7d, the culture solution for respectively obtaining different time points
100 DEG C of water-baths crack 30min, and in -20 DEG C after multigelation 2~3 times, 12000rpm is centrifuged 15min, supernatant is taken to pass through
After 0.22 μm of water phase membrane filtration, Testing and appraisal is carried out using high performance liquid chromatography and triple level four bars mass spectrometer.
Second order ms qualification result shows: the 7th day after substrate addition, in culture solution can stable detection to theanine
(see Fig. 4).Illustrate: the flora being separated to from ten Zhejiang Province Nian Sheng tea tree breeds " Dragon Well tea 43 " of in vitro culture is in vitro culture
Under the conditions of, there is the ability of synthesis theanine.
Embodiment 3
1) four Zhejiang Province Nian Sheng tea tree breeds " black ox is early " tea tree endophyte flora is separately cultured
Superclean bench sterile working: the access Zhejiang Province Pian Siniansheng tea tree breed " black ox is early " sterile tissue culture miaoye of tea tree
Piece, after the sheet of 0.5cm × 1.0cm is cut into scissors, random oblique cutting enters in solid LB media, sealed membrane sealing, 28 DEG C of perseverances
Temperature culture carton upside down culture 2-3 days, after growing flora abundant around blade, is inoculated into LB liquid medium and is carried out
Continue to cultivate.
2) tea tree endophyte group synthesizes the sample preparation and detection process of theanine
By flora in 28 DEG C of shaken cultivations to OD600When being 1.0, final concentration of 20mM paddy is added simultaneously into LB culture solution
Glutamine and 20mM ethamine, and respectively not add the same concentration bacterium solution of any substrate and add the blank culture with concentration of substrate
Base as a control test, under same 28 DEG C of condition of culture, continues after cultivating 7d, the culture solution for respectively obtaining different time points
100 DEG C of water-baths crack 30min, and in -20 DEG C after multigelation 2~3 times, 12000rpm is centrifuged 15min, supernatant is taken to pass through
After 0.22 μm of water phase membrane filtration, Testing and appraisal is carried out using high performance liquid chromatography and triple level four bars mass spectrometer.
Second order ms qualification result shows: the 7th day after substrate addition, in culture solution can stable detection to theanine
(see Fig. 4).Illustrate: the flora being separated to from four Zhejiang Province the Nian Sheng tea tree breeds " black ox is early " of in vitro culture is in vitro culture
Under the conditions of, there is the ability of synthesis theanine.
Embodiment 4
1) three Yunnan Province Nian Sheng tea tree large leafs " cloud is No. 10 anti-" tea tree endophyte flora is separately cultured
Superclean bench sterile working: the access Yunnan Province Pian Sanniansheng tea tree large leaf " cloud is No. 10 anti-" sterile tissue culture of tea tree
Seedling leaf, after the sheet of 0.5cm × 1.0cm is cut into scissors, random oblique cutting enters in solid LB media, sealed membrane sealing, and 28
It DEG C constant temperature incubation carton upside down culture 2-3 days, after growing flora abundant around blade, is inoculated into LB liquid medium
It carries out continuing to cultivate.
2) tea tree endophyte group synthesizes the sample preparation and detection process of theanine
By flora in 28 DEG C of shaken cultivations to OD600When being 1.0, final concentration of 20mM paddy is added simultaneously into LB culture solution
Glutamine and 20mM ethamine, and respectively not add the same concentration bacterium solution of any substrate and add the blank culture with concentration of substrate
Base as a control test, under same 28 DEG C of condition of culture, continues after cultivating 7d, the culture solution for respectively obtaining different time points
100 DEG C of water-baths crack 30min, and in -20 DEG C after multigelation 2~3 times, 12000rpm is centrifuged 15min, supernatant is taken to pass through
After 0.22 μm of water phase membrane filtration, Testing and appraisal is carried out using high performance liquid chromatography and triple level four bars mass spectrometer.
Second order ms qualification result shows: the 7th day after substrate addition, in culture solution can stable detection to theanine
(see Fig. 4).Illustrate: the flora being separated to from three Yunnan Province the Nian Sheng tea tree large leafs " cloud is No. 10 anti-" of in vitro culture is in vitro
Under condition of culture, there is the ability of synthesis theanine.
Claims (8)
1. the flora of one kind production L-thiamine, which is characterized in that the flora is to be separated to from tea tree sterile tissue cultivating seedling
Tea tree endophyte.
2. the flora of production L-thiamine according to claim 1, which is characterized in that partition method are as follows: access piece tea tree group
Seedling leaf is trained, after the sheet of 0.5cm × 1.0cm is cut into scissors, random oblique cutting enters in solid LB media, sealed membrane sealing,
28 DEG C constant temperature incubation carton upside down culture 2-3 days, after growing flora abundant around blade, be inoculated into LB liquid medium
In carry out continuing to cultivate, flora obtained be produce L-thiamine flora.
3. the flora of production L-thiamine according to claim 2, which is characterized in that the tea tree breed is Anhui Province
Tea tree breed " Shu Chazao ", Yunnan Province's tea tree large leaf " cloud anti-No. 10 ", Zhejiang Province's tea tree breed " Dragon Well tea 43 " and " black ox morning ".
4. the flora of production L-thiamine according to claim 1 or 2, which is characterized in that the flora is yellow unit cell
Cordycepps gamboge Bacillus.
5. application of the flora of any of claims 1 or 2 in production L-thiamine.
6. a kind of method for producing L-thiamine, which is characterized in that steps are as follows:
1) the access sterile tissue culture seedling leaf of piece tea tree, after the sheet of 0.5cm × 1.0cm is cut into scissors, random oblique cutting enters solid
In LB culture medium, sealed membrane sealing, 28 DEG C constant temperature incubation carton upside down culture 2-3 days, after growing flora abundant around blade,
It is inoculated into and carries out continuing to cultivate in LB liquid medium；
2) by flora in 28 DEG C of shaken cultivations to OD600When being 1.0, final concentration of 20mM glutamy is added simultaneously into culture solution
Amine and 20mM ethamine cultivate 7d or more；
3) the 100 DEG C of water-baths of culture solution obtained crack 30min, and in -20 DEG C after multigelation 2~3 times, 12000rpm, centrifugation
15min obtains the supernatant for being rich in L-thiamine.
7. the method for production L-thiamine according to claim 7, which is characterized in that in step 2), culture 10 days or more.
8. the method for production L-thiamine according to claim 7, which is characterized in that the tea tree breed is Anhui Province
Tea tree breed " Shu Chazao ", Yunnan Province's tea tree large leaf " cloud anti-No. 10 ", Zhejiang Province's tea tree breed " Dragon Well tea 43 " and " black ox morning ".
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| CN201811545381.6A CN109439596A (en) | 2018-12-17 | 2018-12-17 | The flora of one kind production L-thiamine and its application |
| CN201910207335.3A CN109797122A (en) | 2018-12-17 | 2019-03-19 | The flora of one kind production L-thiamine and its application |
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| CN103409475B (en) * | 2013-07-18 | 2017-03-08 | 江南大学 | A kind of method of enzymatic clarification L theanine |
| CN107164278A (en) * | 2017-06-26 | 2017-09-15 | 湖南农业大学 | One plant of endophytic Bacillus subtilis YC01 and its application in preventing and treating Oil Tea Anthracnose |
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Application publication date: 20190308 |