CN109797122A - 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 PDF

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CN109797122A
CN109797122A CN201910207335.3A CN201910207335A CN109797122A CN 109797122 A CN109797122 A CN 109797122A CN 201910207335 A CN201910207335 A CN 201910207335A CN 109797122 A CN109797122 A CN 109797122A
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flora
thiamine
tea tree
culture
production
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孙俊
常慢慢
宛晓春
张照亮
张正竹
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Anhui Agricultural University AHAU
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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
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
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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
    • 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

The invention discloses the flora of a kind of production L-thiamine and its applications, the flora is the tea tree endophyte being separated to from tea tree sterile tissue cultivating seedling, belongs to Luo Dannuo Bacteriaceae gamboge Bacillus (Rhodanobacteraceae Luteibacter sp.).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 7 days after glutamine and ethamine, experimental group is able to detect that the synthesis of L-thiamine compared with control group, and the theanine synthesized 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

The flora of one kind production L-thiamine and its application
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 70% 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 " 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 flora is Luo Dannuo Bacteriaceae 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 the culture of constant temperature incubation carton upside down 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 1~15d；
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 1), the tea tree breed is Anhui Province " Shu Chazao ", and flora is named as CsEs.
In step 2), preferably culture 7d.
In step 1), the tea tree breed is Yunnan Province tea tree large leaf " cloud is No. 10 anti-", Zhejiang Province's local tea variety " dragon Well 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 7 days after amine, experimental group is able to detect that the synthesis of L-thiamine compared with control group, and is with tea tree breed " Shu Chazao " Example carries out 1~15d of culture, and when discovery cultivates 1~10d after substrate addition, theanine content is persistently increased, the tea in 10d Histidine content average out to 3.6mg/L, hereafter continues to decline.And the theanine of synthesis can rapid secretion to extracellular, secernment efficiency Up to 90% or so, secretion theanine can be efficiently synthesized, there is good practicability.
Detailed description of the invention
Fig. 1 is that flora figure abundant is grown around blade；
Fig. 2 is the liquid chromatography mass spectrometric qualification result figure of " Shu Chazao " tea tree endophyte flora CsEs synthesis theanine；
Fig. 3 is the time plot of " Shu Chazao " tea tree endophyte flora synthesis 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 the culture of constant temperature incubation carton upside down 2~3 days, to leaf It is grown around piece (such as Fig. 1) after flora abundant, 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 3.6mg/L reaches as high as 4.5mg/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 With the ability for efficiently synthesizing theanine.And when cultivating 1~10d after substrate addition, theanine content is persistently increased, the Theanine content average out to 3.6mg/L, hereafter continues to decline when 10d.
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, be inoculated into LB liquid medium into Row continues 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, be inoculated into LB liquid medium into Row continues 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 DEG C the culture of constant temperature incubation carton upside down 2~3 days, after growing flora abundant around blade, it is inoculated into LB liquid medium In carry 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 the culture of constant temperature incubation carton upside down 2~3 days, after growing flora abundant around blade, be inoculated into liquid LB culture It carries out continuing to cultivate in base, flora obtained is the flora for producing L-thiamine.
3. the flora of production L-thiamine according to claim 2, which is characterized in that the tea tree breed is Anhui Province " Shu Chazao ", Yunnan Province's tea tree large leaf " cloud anti-No. 10 ", Zhejiang Province's local tea variety " 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 Luo Dannuo Bacteriaceae 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 the culture of constant temperature incubation carton upside down 2~3 days, to grow flora abundant around blade Afterwards, 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 1~15d；
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 6, which is characterized in that in step 2), cultivate 7d.
8. the method for production L-thiamine according to claim 6, which is characterized in that the tea tree breed is Anhui Province " Shu Chazao ", Yunnan Province's tea tree large leaf " cloud anti-No. 10 ", Zhejiang Province's local tea variety " Dragon Well tea 43 " and " black ox morning ".
CN201910207335.3A 2018-12-17 2019-03-19 The flora of one kind production L-thiamine and its application Pending CN109797122A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115838407A (en) * 2022-11-09 2023-03-24 安徽农业大学 Application of CsHO1 in regulating and controlling accumulation of theanine in tea

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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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CN103409475A (en) * 2013-07-18 2013-11-27 江南大学 Method for synthesizing L-theanine through enzyme process
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115838407A (en) * 2022-11-09 2023-03-24 安徽农业大学 Application of CsHO1 in regulating and controlling accumulation of theanine in tea
CN115838407B (en) * 2022-11-09 2024-04-26 安徽农业大学 Use of CsHO A in regulating theanine accumulation in tea

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Application publication date: 20190524