CN106167784B - A kind of separation and application of D-ALPHA-Hydroxypropionic acid producing strains - Google Patents
A kind of separation and application of D-ALPHA-Hydroxypropionic acid producing strains Download PDFInfo
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- CN106167784B CN106167784B CN201610739714.3A CN201610739714A CN106167784B CN 106167784 B CN106167784 B CN 106167784B CN 201610739714 A CN201610739714 A CN 201610739714A CN 106167784 B CN106167784 B CN 106167784B
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- enterococcus faecium
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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
- C12R2001/01—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
- C12N1/205—Bacterial isolates
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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
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
- C12P7/56—Lactic acid
Abstract
The invention discloses the separation and application of a kind of D-ALPHA-Hydroxypropionic acid producing strains.The D-ALPHA-Hydroxypropionic acid producing strains are enterococcus faecium (Enterococcus faecium) WJ.YT.XY., and collection registration number is CGMCC No.12637.The bacterium can be used for industrial fermentation and produce D-ALPHA-Hydroxypropionic acid, and can develop corresponding industrial chemicals product, research with higher and application value whereby.
Description
Technical field
The present invention relates to microbial fermentation industry, in particular to the separation of one plant D-ALPHA-Hydroxypropionic acid producing strains and the bacterial strain are in D- cream
The application that acid generates.
Background technique
Lactic acid (Lactic acid), scientific name α-hydracrylate or 2 hydroxy propanoic acid.The skeleton symbol of lactic acid is
CH3CHOHCOOH, relative molecular weight 90.It has an asymmetric carbon atom, therefore lactic acid has optical activity, be divided into Pfansteihl and
D-ALPHA-Hydroxypropionic acid.Lactic acid is all very widely used in many fields, such as food, environmental protection, chemical industry, agricultural etc..Human body can be absorbed simultaneously
Using Pfansteihl, but D-ALPHA-Hydroxypropionic acid can not be used, however D-ALPHA-Hydroxypropionic acid obtains good development in other field.It is with D-ALPHA-Hydroxypropionic acid
The fibre that raw material is worked it out has good gas permeability, toughness and wrinkle resistance, can be widely applied to anti-straw bag, farming land
Film, discharge water cloth lid etc., or even the water conservation thin-film material that can also be used as in desert；In terms of medicine, it is used as operation stitching
Line, drug controlled release material, bone renovating material etc.；In terms of clothes product, it can be made knitting, or because of its smoothness and
It is nonirritant, become good underwear raw material.There are mainly three types of production methods for D-ALPHA-Hydroxypropionic acid, are chemical synthesis, micro- life respectively
Object fermentation method and microbial enzyme method.In the comparison of this three, in conjunction with factors such as finished product, safety and environment, in industrialized production
Main selection microbe fermentation method.
At this stage, in order to reduce production cost, production process mainly passes through shortening fermentation period, improves the yield of D-ALPHA-Hydroxypropionic acid
And purity.Research worker both domestic and external is mainly with regard to the optimization of the breeding of D-ALPHA-Hydroxypropionic acid producing bacterial strain, genetic engineering and zymotechnique
These aspects conduct in-depth research.
So far, researchers at home and abroad for the research of D-ALPHA-Hydroxypropionic acid producing strains be concentrated mainly on Sporolactobacillus and
Lactobacillus.Both Pseudomonas can carry out homofermentative lactic using glucose, obtain D-ALPHA-Hydroxypropionic acid by EMP Embden Meyerbof Parnas pathway.By
It is all transition anaerobic bacteria or facultative anaerobic bacteria in them, energy consumption is few, and yield is high, is suitable for large-scale industrial fermentation and produces D- cream
Acid.
Summary of the invention
First purpose of the invention is to provide a kind of bacterial strain that can efficiently produce D-ALPHA-Hydroxypropionic acid.
Strain isolation provided by the present invention for lactic acid producing is from Inner Mongol somewhere soil, with enterococcus faecium
(Enterococcus faecium) bacterial strain similarity up to 99%, is attributed to enterococcus faecium, is named as enterococcus faecium
(Enterococcus faecium)WJ.YT.XY.。
New strains provided by the present invention for production of lactic acid are enterococcus faecium (Enterococcus faecium)
WJ.YT.XY., China Committee for Culture Collection of Microorganisms's common micro-organisms center is preserved on June 17th, 2016
(referred to as CGMCC, address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica), in preservation
Heart registration number is CGMCC No.12637.
The bacterium is gram-positive bacteria, in spherical.There is no bacterium to grow in the fluid nutrient medium that ampicillin is added,
Being added on the fluid nutrient medium of kanamycins has bacterium to grow, and illustrates that bacterial strain does not have resistance to ampicillin, has to kanamycins
Resistance.It can be grown in common bacteria LB screening and culturing medium.It is spherical bacterium under optical microscopy, facultative anaerobic bacteria, can degrade Portugal
Grape sugar produces acid.The bacterium produce D-ALPHA-Hydroxypropionic acid culture medium prescription: glucose 12% (g/mL, similarly hereinafter), peptone 2%, beef extract 1.5%,
Yeast extract 1.5%, sodium acetate 1%, Triammonium citrate 0.4%, dipotassium hydrogen phosphate 0.3%, epsom salt 0.04%, manganese sulfate
0.02%, Tween-80 0.1%.
PH is 6.0~7.0 in the cultural method, and optimum pH is 6.5.
Cultivation temperature is 35~45 DEG C in the cultural method, and optimum temperature is 40 DEG C.
Incubation time is 3~5 days or so in the cultural method.
Enterococcus faecium (Enterococcus faecium) WJ.YT.XY. of the invention can be used for producing D-ALPHA-Hydroxypropionic acid, and can borrow
This develops corresponding industrial chemicals product, research with higher and application value.
The preservation of biomaterial
During enterococcus faecium (Enterococcus faecium) WJ.YT.XY. of the invention was preserved on June 3rd, 2016
State's Microbiological Culture Collection administration committee common micro-organisms center (referred to as CGMCC, address: Chaoyang District, Beijing City North Star west
No. 3 Institute of Microorganism, Academia Sinica, institute of road 1), deposit number is CGMCC No.12637.
Detailed description of the invention
Fig. 1 is that D-ALPHA-Hydroxypropionic acid chemical content measures in enterococcus faecium (Enterococcus faecium) WJ.YT.XY fermentation liquid
As a result；
Fig. 2 is D-ALPHA-Hydroxypropionic acid optics assay in enterococcus faecium (Enterococcus faecium) WJ.YT.XY fermentation liquid
As a result.
Specific embodiment
By the way that the present invention will be described in more detail by following embodiment, it is to be understood that, following embodiment is only illustrative
, the present invention is not limited to these embodiments restrictions.Following experimental methods are conventional method unless otherwise instructed, all cultures
Solvent in base is water.
Enterococcus faecium (Enterococcus faecium) WJ.YT.XY of the invention is in the acquisition of in August, 2015 from China
Mongolian Huhehaote City, autonomous region somewhere soil.
Separation method: strain is to mix growth with various bacterium together under normal operation, and enrichment culture only inhibits
Or those bacteriums for being not suitable with condition of culture have been eliminated, the purebred of bacterium cannot be obtained, it is therefore necessary to be isolated and purified.This
Experiment carries out bacterium using spread plate method (Spread plate method) and plate streak (Streakplate method)
Strain separation.Separation is few by the bacterium solution picking of inorganic salts culture with oese under sterile working using agar plate scribing line separation
Perhaps, it lines on agar plate, under conditions of 40 DEG C, 3~7d is cultivated in biochemical cultivation case, forms bacterium on agar plate
It falls.In order to purify strain more, the separation that drawn lines repeatedly with plating medium is multiple.Choose high concentration region on plating medium
The single colonie of dispersion, then according to the size of bacterium colony, shape, lustrous surface, protuberance degree, transparency, edge shape, bacterium colony
The difference of color finally picks out suitable bacterium colony, it is used oese with sterile working, is seeded in enriched medium, sets
3~7d is cultivated in 40 DEG C of biochemical cultivation case.
Single bacterial strain after purification is seeded on agar medium inclined-plane, 4 DEG C of preservations are raw for recent form and physiology
Change identification to use.
Fermentation process:
1, fermentation medium: glucose 12% (g/mL, similarly hereinafter), peptone 2%, beef extract 1.5%, yeast extract is prepared
1.5%, sodium acetate 1%, Triammonium citrate 0.4%, dipotassium hydrogen phosphate 0.3%, epsom salt 0.04%, manganese sulfate
0.02%, Tween-80 0.1%, pH value 6.2.
2, by the fermentation medium high-temperature sterilization of preparation, the dung intestines of the present invention of stationary phase are inoculated in the fermentation medium later
Coccus carries out Liquid Culture.Inoculation will be carried out aseptically, and aseptic manipulation work table needs to be shone with ultraviolet lamp before use
Sterilizing 30min is penetrated, also needs to carry out disinfection to both hands with alcohol before operation, in operation, it should be noted that station ventilation,
Flame sterilization is carried out to oese before inoculation.The speed 160rmp of shaking table when culture, and the pH value of culture medium is monitored at any time,
It adds calcium carbonate to adjust, the additive amount 4% of calcium carbonate.
3, the bacterium solution for cultivating step 2 to stationary phase carries out amplification fermented and cultured, accesses 25% (V/ in the fermentation medium
V stationary phase bacterium solution) is put into ferment at constant temperature tank and carries out fermentation production D-ALPHA-Hydroxypropionic acid, and producing D-ALPHA-Hydroxypropionic acid temperature is 42 DEG C, fermentation time 3
~5 days.Suitable calcium carbonate is added into fermentor according to the pH value of real time monitoring with real-time pH value monitoring meter for fermentor
Add, makes the pH value in fermentor keep stablizing, prevent yeasting peracid.
4, during the fermentation, the fermentation liquid in fermentor is taken to carry out chromatography prison at interval of certain time (such as 4h)
It surveys, measures the lactic acid production under the time.
Detection method:
1, the chemical content measurement of D-ALPHA-Hydroxypropionic acid uses chromatography in fermentation liquid:
Instrument and condition:
Chromatograph: Shimadzu liquid chromatogram LC-10AT
Detector: wavelength 210nm
Splitter: C18
Mobile phase: 20mM NaH2PO4
Flow: 0.5mL/min
Sample volume: 20 μ L
Sample preparation: fermentation broth sample 1mL is taken to be dissolved to 10mL with flowing phase dilution
Testing result as shown in Figure 1, D-ALPHA-Hydroxypropionic acid appearance time: 6.9min.
2, D-ALPHA-Hydroxypropionic acid optics assay uses chromatography in fermentation liquid:
Instrument and condition:
Chromatograph: Shimadzu liquid chromatogram LC-10AT
Detector: wavelength 254nm
Splitter: SUMICHRAL OA-5000
Mobile phase: 0.002mol/L copper-bath
Flow: 1.2mL/min
Sample volume: 20 μ L
Sample preparation: 1g fermentation fluid samples are weighed, with 0.002mol/L copper sulphate (CuSO4·5H2O) solution is (by de-
Gas) by 100 times of sample dilution, the micro-filtrate membrane filtration for being then 0.2 micron by aperture removes bulky grain that may be present, makes
At measuring samples.
For testing result as shown in Fig. 2, Pfansteihl appearance time is 13.5min, D-ALPHA-Hydroxypropionic acid appearance time is 15.6min.
By the above Analysis of test results, concentration of the D-ALPHA-Hydroxypropionic acid content in fermentation liquid is 15% or so, and chemical content passes through
The means such as rectifying carry out concentration promotion, and the chemical content of concentrate D lactic acid is up to 99%.
Claims (5)
1. a kind of enterococcus faecium (Enterococcus faecium) WJ.YT.XY., collection registration number is CGMCC
No.12637。
2. enterococcus faecium (Enterococcus faecium) WJ.YT.XY. that collection registration number is CGMCC No.12637
A kind of cultural method, cultivated using containing the culture medium of following ingredient: glucose 12%, peptone 2%, beef extract
1.5%, yeast extract 1.5%, sodium acetate 1%, Triammonium citrate 0.4%, dipotassium hydrogen phosphate 0.3%, epsom salt 0.04%,
Manganese sulfate 0.02%, Tween-80 0.1%.
3. cultural method as claimed in claim 2, which is characterized in that cultivation temperature is 35~45 DEG C, incubation time 3~5 days.
4. enterococcus faecium (Enterococcus faecium) WJ.YT.XY. that collection registration number is CGMCC No.12637
Application in D-ALPHA-Hydroxypropionic acid production.
5. application as claimed in claim 4, which is characterized in that enterococcus faecium (Enterococcus faecium)
WJ.YT.XY. it ferments, produces D-ALPHA-Hydroxypropionic acid.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103122325A (en) * | 2012-09-26 | 2013-05-29 | 中国人民解放军总医院 | Space enterococcus faecium LCT (Liquid-based Cytology Test )-EF297 |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103122325A (en) * | 2012-09-26 | 2013-05-29 | 中国人民解放军总医院 | Space enterococcus faecium LCT (Liquid-based Cytology Test )-EF297 |
Non-Patent Citations (3)
| Title |
|---|
| Enterococcus faecium QU50:a novel thermophilic lactic acid bacterium for high-yield L-lactic acid production from xylose.;Mohamed Ali Abdel-Rahman et al.;《FEMS Microbiology letters》;20150101;第362卷(第2期);第1-7页 * |
| The microflora of the sour dough of wheat flour bread Ⅻ.Effect of freezing on the viability and functional properties in wheat flour doughs of microbial mass from lactic acid bacteria;M.José Torner et al.;《Zeitschrift für Lebensmittel-untersuchung and Forschung》;19891231;第189卷(第6期);表3 * |
| 新疆传统发酵酸乳中屎肠球菌Enterococcus Faecom的高密度培养;胡敏等;《中国乳品工业》;20120430;第40卷(第4期);第12-17页 * |
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Effective date of registration: 20190726 Address after: 238251 Anxing Road, Anhui Fine Chemical Industry Base, Wujiang Town, Ma'anshan City and County, Anhui Province Applicant after: Anhui Anhe Biotechnology Co., Ltd. Address before: 238101 Gulou Community Health Lane 2-2, Huanfeng Town, Hanshan County, Ma'anshan City, Anhui Province Applicant before: Cheng Jingzheng |
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