CN104342377A - Zygosaccharomyces rouxii strain for producing D-arabitol and application thereof - Google Patents
Zygosaccharomyces rouxii strain for producing D-arabitol and application thereof Download PDFInfo
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- CN104342377A CN104342377A CN201410337503.8A CN201410337503A CN104342377A CN 104342377 A CN104342377 A CN 104342377A CN 201410337503 A CN201410337503 A CN 201410337503A CN 104342377 A CN104342377 A CN 104342377A
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- C—CHEMISTRY; METALLURGY
- 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
- C12N1/14—Fungi; Culture media therefor
- C12N1/145—Fungal isolates
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
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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
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
- C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
- C12P7/18—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic polyhydric
Abstract
The invention discloses a zygosaccharomyces rouxii strain for producing D-arabitol and application thereof, and relates to the fields of development and application of a microorganism industrial strain. The hypertonic resistant yeast zygosaccharomyces rouxii for producing D-arabitol is separated from natural chaste honey. The strain disclosed by the invention is preserved in China Center For Type Culture Collection with preservation No. of CCTCC NO: M 2014205. 5L fermentation tank test tracking shows that the strain is normal in metabolism and stable in fermentation performance. The yeast strain is used for producing D-arabitol by converting substrate D-glucose, which is simple in process and capable of realizing conservation and environmental protection, and a conversion rate can reach 40%; the strain has practical application value, and is an excellent yeast strain for producing D-arabitol.
Description
Technical field
The present invention relates to the exploitation of microbiological industry bacterial strain and Application Areas, relate to a kind of produce D-R alcohol yeast bacterium screening and convert D-glucose produce the technological method of D-R alcohol.
Background technology
D-R alcohol is a kind of naturally occurring five carbon multi-sugar alcohols, also known as D-(+)-arabitol, D(+)-gummy sugar alcohol, its molecular formula is C
5h
12o
5, be isomers with xylulose and Xylitol.Under normal temperature, D-R alcohol sterling outward appearance is white crystal, and pleasantly sweet, water absorbability is strong.D-R alcohol is a kind of natural low caloric value sweet taste substance having better nutritivity and be worth, anti-dental caries, metabolism in living organism does not affect insulin level, can be used as the nutrition agent of diabetics, therapeutical agent, children's preventing decayed tooth food, foodstuff additive are also a kind of synthetic intermediates of important nervosa medicine simultaneously.Therefore, D-R alcohol is at food, and chemical industry, medicine and other fields has important using value.
At present, the preparation method of D-R alcohol mainly adopts chemosynthesis and biological synthesis process.Namely chemical synthesis is obtained by catalytic reduction D-R alcohol or D-lyxose, this method complex process, and equipment requirements is high, and environmental pollution is serious, not yet accomplishes scale production.Biological synthesis process utilizes the enzyme system in microbe to be D-R alcohol by conversion of glucose, and production technique is simple, operates extensive, and it is large to invest little income.The people such as Spencer find that osmophilic yeast can produce polyvalent alcohol the earliest, open the new way of producing multi-sugar alcohol.It is feasible that the people such as Onishi report transforming glucose production D-R alcohol.Subsequently, the research utilizing microorganism to prepare D-R alcohol receives the extensive concern of Chinese scholars.
The principal element of current restriction D-R alcohol output is generation and the low-conversion of by product, is the method obtaining production starting strain of most economical practicality from the quality yeast of physical environment direct separation screening high yield D-R alcohol.The present invention be separated to from natural twigs of the chaste tree nectar sample resistance to height that a strain produces D-R alcohol ooze Lu Shi zygosaccharomyces (
zygosaccharomyces rouxii).The bacterial strain that provides is preserved in China typical culture collection center, and deposit number is: CCTCC M 2014205.Utilize this yeast strain conversion of substrate D-Glucose to produce D-R alcohol, technique is simple, economical environment-protective, and its transformation efficiency can reach 40%, has actual application value, is the excellent product D-R alcohol yeast bacterial strain of a strain.
Summary of the invention
The resistance to height that technical problem to be solved by this invention is to provide the new product D-R alcohol of a strain oozes methamidophos strain, and the application in glucose fermentation.
The present invention first utilizes hypertonic liquid substratum to carry out enrichment culture to gathered natural nectar sample, coat height subsequently and ooze flat board, the doubtful bacterium colony of picking carries out shake flask fermentation, adopt thin layer (Microcrystalline Cellulose) chromatography to carry out initial characterization to the glycitols material in fermented liquid to detect and thick detection by quantitative, preliminary screening goes out other by products such as D-R alcohol output is high, glycerine and yields poorly and the bacterial strain of glucose less residue, its fermented liquid is carried out quantitatively screening again by high performance liquid chromatography again survey, the yeast strain that seed selection is excellent.
1, Lu Shi zygosaccharomyces bacterial strain JM-C46(provided by the present invention
zygosaccharomyces rouxiijM-C46), be preserved on May 14th, 2014 China typical culture collection center being positioned at Wuhan, China Wuhan University, deposit number is: CCTCCNO:M 2014205.
2, the present invention utilizes provided yeast strain, 250ml triangular flask is adopted to carry out shake flask fermentation, first be optimized by the fermention medium component of response phase method (RSM) to test strain conversion of substrate glucose, then by single factor experiment, fermentation condition be optimized; Finally adopt the test of 5L fermentor tank, evaluate the leavening property of this bacterial strain further and produce the ability of D-R alcohol.
Fermentation test result shows: this bacterial strain glucose-tolerant concentration can reach 500g/L.The most adaptable method is: substrate D-Glucose concentration 20-25%, yeast extract 1.07%, Tryptones 0.72%, leavening temperature 30 DEG C, natural pH, shaking speed 200rpm, transformation time 4 days, inoculum size 10%.
5L fermentor tank test-results shows: this bacterial strain is after the most adaptable method bottom fermentation terminates: glucose residual is: 3.89 g/L, D-R alcohol content are: 79.16 g/L, glycerol content are: 9.43 g/L, ethanol content are: 3.21g/L.The product D-R alcohol ability of this bacterial strain is higher.
Beneficial effect:
1) the present invention has adopted high sugared enrichment culture technique, thin-layer chromatography technology and high-efficient liquid phase chromatogram technology to screen to produce the strain excellent CCTCC M 2014205 of D-R alcohol.The ability that this bacterial strain produces D-R alcohol is higher.Certain promoter action is had to the technological line of development bio-transformation glucose production D-R alcohol.
2) property indices that fermentor tank test is followed the tracks of shows: this bacterial strain Metabolism of Normal, and it is strong to produce D-R alcohol ability, and the by-products content such as glycerine, ethanol is lower, and glucose residual sugar amount is lower, is that bacterial strain produced by the excellent D-R alcohol of a strain.
Embodiment
The following examples can make this area researchist more fully understand the present invention, but do not limit the present invention in any way.
1, Lu Shi zygosaccharomyces bacterial strain JM-C46(provided by the present invention
zygosaccharomyces rouxiijM-C46), be preserved on May 14th, 2014 China typical culture collection center being positioned at Wuhan, China Wuhan University, deposit number is: CCTCCNO:M 2014205.
This bacterial strain feature is as follows:
Examine under a microscope, the cell of this bacterial strain is circular, multiterminal budding; On solid medium, this bacterial strain colony characteristics is: cream-colored, and protruding, smooth surface is moistening, thickness, neat in edge, and bacterium colony is less.
Yeast strain of the present invention adopts following flow process to carry out seed selection:
Nectar sample collecting, hypertonic liquid substratum enrichment culture, gradient dilution, coating be high oozes flat board, line is separated doubtful bacterium colony, carry out shake-flask culture, thin layer chromatography primary dcreening operation, high performance liquid chromatography are sieved again, the test of 5L fermentor tank, obtain object bacterial strain, qualification, preservation.
Embodiment 1:
Specific operation process is as follows:
1, height oozes enrichment culture
Preparation enrichment medium: 40% D-glucose, 1% yeast extract, 1% peptone.
Different nectar sample takes 1.0 g respectively, and join in the 250ml triangular flask containing 20ml enrichment medium, vortex is even.30 DEG C, 200r/min, shake-flask culture 3d.
After final enrichment culture thing gradient dilution, draw 200 μ l respectively and be applied to height and ooze flat board, cultivate 3d for 30 DEG C.
Select well-grown doubtful yeastlike single bacterium colony line separation and purification, inclined-plane is stored in 4 DEG C of refrigerators.
2, TLC method primary dcreening operation is adopted
Preparation fermention medium: 20% D-glucose, 1% yeast extract, 1% peptone.
At Bechtop, by the different yeast strains be separated to, respectively get in the 250ml triangular flask that an articulating enters containing 20ml fermention medium.30 DEG C, 200r/min, shake-flask culture 4d.
Fermentation liquor treatment method: the centrifugal 10min of fermented liquid each 1ml, 10000r getting different strains.
With Microcrystalline Cellulose plate for chromatography media, developping agent and proportioning thereof are: ethyl acetate: pyridine: Glacial acetic acid: water=16:10:2:3.Before chromatography, chromatography agent needs presaturation 1h.
The mixed sample of preparation 2%D-glucose and 2%D-arabitol standardized solution and the two equal proportion, chromatography applied sample amount is 1 μ L, and some line-transect is positioned at distance chromatoplate lower rim 1.8 centimeters, point sample dries after terminating, can put into chromatography cylinder, the chromatography time is 80min, repeats chromatography 3 ~ 4 times.
Chromatography terminates naturally to dry afterwards, or is placed in the evaporation that remaining reagent accelerated by 50 DEG C of baking ovens.
The preparation of developer: Silver Nitrate acetone soln A-saturated sodium hydroxide spirituous solution B
A: get the saturated silver nitrate solution of 0.1mL, by acetone diluted to 20mL, more dropwise add water to nitric acid
Silver dissolves; B: NaOH saturated aqueous solution alcohol is diluted to 0.5mol/L.
First Silver Nitrate-acetone soln is evenly sprayed on chromatoplate with triangle watering can, after fully drying, sprays sodium hydroxide-ethanolic soln again.
Mark the bacterial strain that D-R alcohol content is higher after colour developing, wait for follow-up further screening.
3, HPLC method is adopted to sieve again
Preparation fermention medium: 20% D-glucose, 1% yeast extract, 1% peptone.
At Bechtop, the yeast strain obtained by primary dcreening operation, respectively gets in the 250ml triangular flask that an articulating enters containing 20ml fermention medium.30 DEG C, 200r/min, shake-flask culture 4d.
Fermentation liquor treatment method: each 1ml of fermented liquid getting different strains, with the centrifugal 20min of isopyknic trichloroacetic acid precipitation albumen 3h, 12000r, through 0.45 μm of water film filtering process, sample size 20 μ L.
The preparation of 1% mass concentration standardized solution: accurately take D-Glucose, D-R alcohol, erythritol, ethanol, each 0.1g of glycerine, with acetonitrile solution (V
acetonitrile: V
water=1:1) be settled to 10ml.
The analysis condition of sample determination: adopt differential refraction detector (RID, ShodexRI100), chromatographic column model:, column temperature 35 DEG C, moving phase is acetonitrile solution (V
acetonitrile: V
water=80:20), flow velocity 0.8mL/min, detector temperature 35 DEG C, pump pressure 43bar.
Sample determination method: open detector, preheating makes to stablize; Setting routine analyzer; Chromatographic column is installed, opens liquid chromatograph, pass into moving phase, setting flow velocity 0.1mL/min, balance 2h; Make moving phase flow through sample pool, after baseline stability, at the input of sample table sample message, by setup program test sample, carry out the concentration of assay products and substrate according to identical retention time.
4, shake flask fermentation test
1) go bail on Bechtop yeast one ring be hidden on test tube slant, access is equipped with 100ml and is sent out
In the 500ml triangular flask of ferment substratum, 30 DEG C, 200rpm cultivates 16h, in this, as seed fermentation liquid.
2) optimization of glucose concn
Configure the fermention medium 50ml of different glucose concn (15,20,25,30,35,40%) in 250ml triangular flask, other composition is identical, comprises 1% yeast extract, 1% peptone.Access with inoculum size 5% in each substratum configured, 30 DEG C, 200rpm, cultivation is after 4 days, the centrifugal 10min of fermented liquid 10000r, utilize TLC method and HPLC method to detect residual sugar amount in fermented liquid supernatant and D-R alcohol amount.
3) optimization of inoculum size
Transfer in fermention medium (glucose concn 25%) with different vaccination amount (5,8,10,12,15%) by seed fermentation liquid, 30 DEG C, 200rpm, cultivation is after 4 days, the method detecting residual sugar amount and D-R alcohol amount in fermented liquid supernatant is the same.
4) determination of culture temperature
With optimum inoculation amount (10%), seed fermentation liquid is transferred in fermention medium (glucose concn 25%), under different culture temperature (25,28,30,33,37 DEG C) condition, after 200rpm cultivates 4 days, the method detecting residual sugar amount and D-R alcohol amount in fermented liquid supernatant is the same.
5) determination of incubation time
With optimum inoculation amount (10%), seed fermentation liquid is transferred in fermention medium (glucose concn 25%), 30 DEG C, 200rpm cultivates 6 days, sample 1ml every 24h, the method detecting residual sugar amount and D-R alcohol amount in different times fermented liquid supernatant is the same.
6) determination of shaking speed
With optimum inoculation amount (10%), seed fermentation liquid is transferred in fermention medium (glucose concn 25%), different rotating speeds (100,150,200,250rpm) under, after 30 DEG C of cultivation 96h, the method detecting residual sugar amount and D-R alcohol amount in fermented liquid supernatant is the same.
5,5L fermentor tank test
1) go bail on Bechtop yeast one ring be hidden on test tube slant, access is equipped with in the 500ml triangular flask of 100ml fermention medium, 30 DEG C, 200rpm cultivates 16h, makes thalline be in mid log phase.
2) the bacterial classification access being in logarithmic phase is equipped with in the 5L fermentor tank of 2L fermented liquid.Inoculum size is 10%, 30 DEG C, 200rpm cultivates 4-6 days, and holding oxygen control 10%(Ventilation Rate whole vegetative period is 0.5L/min).
3) after fermentation ends: glucose residual is: 3.89 g/L, D-R alcohol content are: 79.16 g/L, glycerol content are: 9.43 g/L, ethanol content are: 3.21 g/L.The ability that this bacterial strain produces D-R alcohol is higher, and transformation efficiency can reach 40%.
Claims (2)
1. Lu Shi zygosaccharomyces bacterial strain JM-C46
zygosaccharomyces rouxiijM-C46, deposit number is: CCTCCNO:M 2014205.
2. the application of Lu Shi zygosaccharomyces bacterial strain according to claim 1, carry out according to following step:
1) go bail on Bechtop yeast one ring be hidden on test tube slant, access is equipped with 100ml and is sent out
In the 500ml triangular flask of ferment substratum, 30 DEG C, 200rpm cultivates 16h, in this, as seed fermentation liquid;
2) optimization of glucose concn
Configuration glucose concn be the fermention medium 50ml of 15-40% in 250ml triangular flask, other composition is identical, and comprising mass concentration is 1% yeast extract, and mass concentration is 1% peptone; Access with inoculum size 5% in each substratum configured, 30 DEG C, 200rpm, cultivation is after 4 days, the centrifugal 10min of fermented liquid 10000r;
3) optimization of inoculum size
Take volume ratio as 5-15% inoculum size seed fermentation liquid is transferred glucose quality concentration be in 25% fermention medium, 30 DEG C, 200rpm, cultivation is after 4 days;
4) determination of culture temperature
Be 10% inoculum size seed fermentation liquid transferred glucose quality concentration with volume ratio be in 25% fermention medium in fermention medium, under culture temperature 25-37 DEG C of condition, after 200rpm cultivates 4 days;
5) determination of incubation time
Be 10% inoculum size seed fermentation liquid transferred glucose quality concentration with volume ratio be in 25% fermention medium in fermention medium, 30 DEG C, 200rpm cultivates 6 days, sample 1ml every 24h, the method detecting residual sugar amount and D-R alcohol amount in different times fermented liquid supernatant is the same;
6) determination of shaking speed
Be 10% inoculum size seed fermentation liquid transferred glucose quality concentration with volume ratio be in 25% fermention medium in fermention medium, under 100-250rpm rotating speed, 30 DEG C cultivate 96h after,
7) 5L fermentor tank test
A. go bail on Bechtop yeast one ring be hidden on test tube slant, access is equipped with in the 500ml triangular flask of 100ml fermention medium, 30 DEG C, 200rpm cultivates 16h, makes thalline be in mid log phase;
B. the bacterial classification access being in logarithmic phase is equipped with in the 5L fermentor tank of 2L fermented liquid;
Inoculum size is 10%, 30 DEG C, 200rpm cultivates 4-6 days, and hold oxygen whole vegetative period and control 10%, Ventilation Rate is 0.5L/min;
C. after fermentation ends: glucose residual is: 3.89 g/L, D-R alcohol content are: 79.16 g/L, glycerol content are: 9.43 g/L, ethanol content are: 3.21 g/L; The ability that this bacterial strain produces D-R alcohol is higher, and transformation efficiency can reach 40%.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112730694A (en) * | 2021-01-28 | 2021-04-30 | 成都第一制药有限公司 | A herba Leonuri injection with controlled D-arabitol content and its quality control method |
| CN113030359A (en) * | 2021-01-28 | 2021-06-25 | 成都第一制药有限公司 | Detection method for various index components in motherwort injection and quality control method of motherwort injection |
| CN115322910A (en) * | 2022-04-18 | 2022-11-11 | 江苏大学 | Saccharomyces rouxii and mutagenesis screening method and application thereof |
Citations (2)
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| US5631150A (en) * | 1992-11-05 | 1997-05-20 | Xyrofin Oy | Manufacturing of xylitol using recombinant microbial hosts |
| CN101285082A (en) * | 2008-05-28 | 2008-10-15 | 南京工业大学 | Process for producing eutrit by waste xylose mother liquor or eutrit mother liquor in production process of eutrit |
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2014
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5631150A (en) * | 1992-11-05 | 1997-05-20 | Xyrofin Oy | Manufacturing of xylitol using recombinant microbial hosts |
| CN101285082A (en) * | 2008-05-28 | 2008-10-15 | 南京工业大学 | Process for producing eutrit by waste xylose mother liquor or eutrit mother liquor in production process of eutrit |
Non-Patent Citations (2)
| Title |
|---|
| BADAL C等: "Production of D-arabitol by a newly isolated Zygosaccharomyces rouxii", 《J IND MICROBIOL BIOTECHNOL》 * |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112730694A (en) * | 2021-01-28 | 2021-04-30 | 成都第一制药有限公司 | A herba Leonuri injection with controlled D-arabitol content and its quality control method |
| CN113030359A (en) * | 2021-01-28 | 2021-06-25 | 成都第一制药有限公司 | Detection method for various index components in motherwort injection and quality control method of motherwort injection |
| CN115322910A (en) * | 2022-04-18 | 2022-11-11 | 江苏大学 | Saccharomyces rouxii and mutagenesis screening method and application thereof |
| CN115322910B (en) * | 2022-04-18 | 2024-04-09 | 江苏大学 | Russell yeast and mutagenesis screening method and application thereof |
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