CN109401993A - A kind of D-ALPHA-Hydroxypropionic acid production bacterial strain and the method using bacterial strain production D-ALPHA-Hydroxypropionic acid - Google Patents
A kind of D-ALPHA-Hydroxypropionic acid production bacterial strain and the method using bacterial strain production D-ALPHA-Hydroxypropionic acid Download PDFInfo
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Abstract
The present invention relates to a kind of D-ALPHA-Hydroxypropionic acid production bacterial strain and utilize the method for D-ALPHA-Hydroxypropionic acid production bacterial strain production D-ALPHA-Hydroxypropionic acid.When using D-ALPHA-Hydroxypropionic acid production bacterial strain COLmDH production D-ALPHA-Hydroxypropionic acid of the invention, acid production speed and product optical purity in D-ALPHA-Hydroxypropionic acid production are all significantly improved, that is, D-ALPHA-Hydroxypropionic acid production bacterial strain COLmDH of the invention has the product acid activity of the production D-ALPHA-Hydroxypropionic acid significantly improved.Correspondingly, fermentation production of D-lactic acid is come by using D-ALPHA-Hydroxypropionic acid production bacterial strain COLmDH of the invention, the production of D-ALPHA-Hydroxypropionic acid can be substantially improved.Meanwhile D-ALPHA-Hydroxypropionic acid production bacterial strain COLmDH also has the advantage that fermentation costs are low, environmental-friendly.
Description
Technical field
The present invention relates to genetic engineering fields and field of microbial fermentation.It can be fast specifically, the present invention relates to one kind
Fast-growing produces the D-ALPHA-Hydroxypropionic acid production bacterial strain of high optical purity D-lactic acid and the side using D-ALPHA-Hydroxypropionic acid production bacterial strain production D-ALPHA-Hydroxypropionic acid
Method.
Background technique
Polylactic acid (Polylactic acid, PLA) is high score biodegradable made of a kind of polycondensation as lactic acid monomer
Sub- polymer is mainly obtained as monomer by polymerization reaction using the tunning Pfansteihl of microorganism.PLA is because of its raw material
For reproducible living resources, new century most promising novel " bio-based materials " are unanimously regarded as by industrial circle.This
Outside, the product glossiness made of PLA, the transparency, feel and heat-resist also have certain fungus resistance, anti-flammability and anti-
It is UV protection, and glossiness with higher and processing performance；PLA also has nontoxic, nonirritant and good biocompatibility
The features such as.Therefore, PLA has a vast market foreground, and purposes is very extensive, is currently used primarily in clothes, building, agricultural, woods
In the multiple fields such as industry, papermaking and health care.
As that studies PLA deepens continuously, many researchers both domestic and external have found pure poly (l-lactic acid) (PLLA)
Multiple performance is unstable, and the D-ALPHA-Hydroxypropionic acid that proper proportion is added carry out polymerization its performance can be improved, widened answering for polylactic acid
Use range.For example, PLLA is limited in some applications due to the disadvantages of property is crisp, impact resistance is poor and thermal instability
System, in this regard, the copolymerization of high molecular weight can be obtained if a certain proportion of D-ALPHA-Hydroxypropionic acid and Pfansteihl are carried out combined polymerization by control
To reinforce the mechanical performance of polylactic acid, such as impact resistance, tensile strength and ductility etc., while PLLA also can be improved in object
Thermal stability, accelerate the degradation speed of PLLA.On the other hand, D-ALPHA-Hydroxypropionic acid of the optical purity greater than 99% is in medicine, pesticide, change
Work etc., which has, to be widely applied.Generalization can be substituted using high optical voidness D-ALPHA-Hydroxypropionic acid as poly-lactic acid material made of polymerizable raw material
Plastics that chemical product is polymerized, fibre, are applied in medical bone inside-fixture, cigarette filter head, weaving high fiber
It etc. high-end consumer field, while being also the main raw material(s) of 3D printing technique.
So far, D-ALPHA-Hydroxypropionic acid can mainly be obtained by chemical synthesis and microbe fermentation method.Chemical synthesis exists
Environmental pollution, expensive, technology is complicated, the more low thorny problem of optical purity, it is difficult to meet application request.Compared to it
Under, microbe fermentation method is that raw material produces D-ALPHA-Hydroxypropionic acid using renewable resources such as glucose, low, the product optics with production cost
The advantages that purity and highly-safe, working condition are mild, pollution is small, therefore, current global D-ALPHA-Hydroxypropionic acid industrial production are big absolutely
Part is carried out by microbe fermentation method.However, most lactic acid bacteria can only produce Pfansteihl and D-ALPHA-Hydroxypropionic acid simultaneously, only
There are the natural lactic acid bacteria of only a few and some mode engineering bacterias to can be used for specially producing D-ALPHA-Hydroxypropionic acid.The D-ALPHA-Hydroxypropionic acid fermentation being currently known
The production bacterial strain used in production has Lactobacillus delbrueckii (Lactobacillus delbrueckii), lactobacillus bulgaricus
(Lactobacillus bulgaricus) and D-lactic acid bacillus (Bacillus laevolacticus) etc..
However, selectable bacterial strain still extremely has when passing through microbe fermentation method large-scale industrial production D-ALPHA-Hydroxypropionic acid
Limit, needs further breeding high-yield bacterial strain, constantly excavation novel bacterial, to realize yield increases, purity improves, cost reduces,
The targets such as benefit raising.
Summary of the invention
It is a primary object of the present invention to overcome the deficiencies in the prior art described above, bloom can quickly be produced by providing one kind
Learn the D-ALPHA-Hydroxypropionic acid production bacterial strain of purity D-lactic acid and the method using D-ALPHA-Hydroxypropionic acid production bacterial strain production D-ALPHA-Hydroxypropionic acid.It is specific and
Speech, be currently known Leuconostoc mesenteroides (Leuconostoc mesenteroides) can fermenting carbohydrate generate it is a variety of acid and alcohol,
With abilities such as high acid ability, oxidation resistance and antagonism pathogenic bacteria, however, for producing height using Leuconostoc mesenteroides
The D-ALPHA-Hydroxypropionic acid of optical purity, the rare report in this field.In this regard, the present invention is by carrying out the Leuconostoc mesenteroides for being isolated from Yoghourt
Atmospheric pressure at room plasma (atmospheric and room temperature plasma, ARTP) mutagenesis, screening obtain
D-ALPHA-Hydroxypropionic acid speed of production is fast and the bacterial strain of product D-ALPHA-Hydroxypropionic acid optical purity high (up to 99.4%), so as to complete the present invention.
Therefore, according to first aspect, the present invention provides D-ALPHA-Hydroxypropionic acids to produce bacterial strain COLmDH, and the D-ALPHA-Hydroxypropionic acid produces bacterium
The specific name of strain COLmDH was Leuconostoc mesenteroides (Leuconostoc mesenteroides), on December 26th, 2017
It is preserved in (the BeiChen West Road, Chaoyang District, BeiJing City 1 China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC)
Number institute 3, Institute of Microorganism, Academia Sinica), deposit number is CGMCC No.15131.
According to the second aspect, the present invention provides the sides using D-ALPHA-Hydroxypropionic acid production bacterial strain COLmDH production D-ALPHA-Hydroxypropionic acid
Method, which comprises the D-ALPHA-Hydroxypropionic acid production bacterial strain COLmDH is cultivated in the medium, to generate in the medium
With accumulation D-ALPHA-Hydroxypropionic acid；And D-ALPHA-Hydroxypropionic acid is collected from the culture medium.
Beneficial effect
Compared to wild type Leuconostoc mesenteroides, when using D-ALPHA-Hydroxypropionic acid production bacterial strain COLmDH production D-ALPHA-Hydroxypropionic acid of the invention,
Acid production speed and product optical purity are all significantly improved, that is, D-ALPHA-Hydroxypropionic acid production bacterial strain COLmDH of the invention has significant
The product acid activity of the production D-ALPHA-Hydroxypropionic acid of raising.Correspondingly, it is fermented by using D-ALPHA-Hydroxypropionic acid production bacterial strain COLmDH of the invention
D-ALPHA-Hydroxypropionic acid is produced, the production of D-ALPHA-Hydroxypropionic acid can be substantially improved.Meanwhile D-ALPHA-Hydroxypropionic acid production bacterial strain COLmDH also has fermentation costs
Low, environmental-friendly advantage.
Other feature and advantage of the invention will be described in detail by following specific embodiments.
Detailed description of the invention
Fig. 1 is to show to utilize tunning D-ALPHA-Hydroxypropionic acid when D-ALPHA-Hydroxypropionic acid production bacterial strain COLmDH production D-ALPHA-Hydroxypropionic acid of the invention
Optical purity (99.4%) chromatogram.
Specific embodiment
Detailed description of the preferred embodiments below.It should be understood that described herein specific
Embodiment is merely to illustrate and explain the present invention, and is not intended to restrict the invention.
Terms used herein " increase ", " raising ", " enhancing " or " activation " usually can mean that statistically significant amount
Increase.However, to avoid doubt, reference level (such as open country is compared in term " increases ", " raisings ", " enhancing " or " activation " expression
Level in raw type Leuconostoc mesenteroides bacterial strain) increase at least 10%, such as increase at least about 20% or at least about 30% or
At least about 40% or at least about 50% or at least about 60% or at least about 70% or at least about 80% or at least about 90%,
Or increase any amount between 10% to 100% up to and including increase by 100% or compared to reference level；Or compare reference
Horizontal at least about 2 times or at least about 3 times or at least about 4 times or at least about 5 times or at least about 10 times of increase or at 2 times
And the increase or a greater amount of increases of any amount between 10 times.
High optical purity D-lactic acid can be quickly produced in order to filter out in the microorganism for belonging to Leuconostoc mesenteroides
D-ALPHA-Hydroxypropionic acid produces bacterial strain, can be out of office by conventional mutagenesis techniques and low energy ion beam implantation induced-mutation technique or genetic engineering technology etc.
Mutation or variation are introduced in raw type Leuconostoc mesenteroides bacterial strain.The example of induced-mutation technique includes for example being lured using low energy ion beam implantation
The method of change, ion beam as a kind of novel mutation source in terms of mutation breeding due to its unique mutagenic mechanism and biological effect
Develop extremely rapid.Compared with classic mutagenesis source, ion implanting other than with energy sedimentary effect, also have kinetic energy transmitting,
The neutralization of quality deposition and charge and exchange effect etc..It rolls into one physical mutagenesis and chemical mutagenesis characteristic, can be low
In the case that dosage injection, cellular damage are relatively light, induce the change of the basic unit base of inhereditary material or induce chromosome knob
The variation of structure.The example of induced-mutation technique further includes the method for example using X-ray or ultraviolet radiation, or is tried using mutagenesis
The method of agent such as N- methyl-N '-nitro-N nitrosoguanidine and such processing.
In a preferred embodiment of the invention, atmospheric pressure at room plasma (atmospheric and room is utilized
Temperature plasma, ARTP) mutagenesis is transformed Leuconostoc mesenteroides, so that bloom can quickly be produced by filtering out
The D-ALPHA-Hydroxypropionic acid for learning purity D-lactic acid produces bacterial strain.ARTP has been developed in recent years a kind of plasma source, can be in atmosphere
It is (former including helium atom, oxygen atom, nitrogen in excitation state with high activity particle between 25-40 DEG C that pressure generates temperature
Son, OH free radical etc.) concentration plasma jet.Active particle in plasma acts on microorganism, can make micro- life
Object cell wall/film structure and permeability changes, and cause gene damage, and then make microbial gene sequences and its metabolism network
Significant changes eventually lead to microorganism and generate mutation.
In the method for fermentation production of D-lactic acid of the invention, D-ALPHA-Hydroxypropionic acid production bacterial strain COLmDH of the invention is sent out
Ferment culture can obtain D-ALPHA-Hydroxypropionic acid.Wherein, the method for fermented and cultured can be the fermentation that this field is produced conventionally used for D-ALPHA-Hydroxypropionic acid
Method.Using the standard culture procedures of Leuconostoc mesenteroides prepare the seed liquor of D-ALPHA-Hydroxypropionic acid of the present invention production bacterial strain COLmDH with
Fermentation liquid.For example, the method for fermented and cultured, which can comprise the following steps that, produces bacterial strain or cryopreservation for freshly prepared D-ALPHA-Hydroxypropionic acid
D-ALPHA-Hydroxypropionic acid production bacterial strain (such as frozen in glycerol cryopreservation tube in such as -80 DEG C of refrigerators D-ALPHA-Hydroxypropionic acid production bacterial strain) inoculation
It is activated in Leuconostoc mesenteroides fluid nutrient medium；It is incubated overnight obtained seed liquor；Above-mentioned seed liquor is seeded to D- cream
Expand culture in sour acidogenic fermentation culture medium (such as in shaking flask equipped with D-ALPHA-Hydroxypropionic acid acidogenic fermentation culture medium or fermentor) to be made
Fermentation liquid.
For example, the seed liquor can be prepared by following process: picking from the plate the inoculation of Leuconostoc mesenteroides single colonie
In seed liquid culture medium, cultivate 12 under 35-40 DEG C (preferably 37 DEG C) with revolving speed 120-180rpm~for 24 hours to get seed liquor.
In a preferred embodiment of the invention, the seed liquid culture medium is MRS fluid nutrient medium.
For example, the fermentation liquid can be prepared by following process: being inoculated with seed liquor with the inoculum concentration of 5%-10% volume
Into the acidogenic fermentation culture medium being placed in such as shaking flask or fermentor, with revolving speed 120-180rpm at 35-40 DEG C (preferably 37
DEG C) under continuously ferment 72-96h to get fermentation liquid.Wherein, the acidogenic fermentation culture medium include glucose, yeast extract,
Sodium acetate, phosphate, microelement and neutralizer.In a preferred embodiment of the invention, the acidogenic fermentation culture medium
Include following ingredient: glucose 80-160g/L, yeast extract 10g/L, sodium acetate 2g/L, KH2PO4 0.5g/L、MgSO4·
7H2O 0.5-1g/L、MnSO40.1-0.2g/L, Tween 80 1ml/L and suitable neutralizer.It is more excellent in the present invention one
In the embodiment of choosing, the neutralizer is CaCO3, but the present invention is not limited thereto.In a particularly preferred reality of the invention
It applies in mode, the acidogenic fermentation culture medium includes following ingredient: glucose 80-150g/L, yeast extract 10g/L, acetic acid
Sodium 2g/L, KH2PO4 0.5g/L、MgSO4·7H2O 0.5-1g/L、MnSO40.1-0.2g/L, Tween 80 1ml/L and
CaCO3 40-80g/L。
After completing fermented and cultured, the D-ALPHA-Hydroxypropionic acid accumulated in acidogenic fermentation culture medium can be collected by known method.Example
Such as, can by be included in remove cell after acidogenic fermentation culture medium is concentrated so that product crystallization, ion-exchange chromatography and such as
Such method separates D-ALPHA-Hydroxypropionic acid.
It, can also be by known method to the D- cream accumulated in acidogenic fermentation culture medium meanwhile after completing fermented and cultured
Sour or separated D-ALPHA-Hydroxypropionic acid is detected.For example, can be by high performance liquid chromatography and suchlike method come to D-
The yield and optical purity of lactic acid are detected.
Embodiment
Next, by following embodiment, the present invention will be described in more detail, but these embodiments are merely to illustrate
The present invention and be not intended to limit the scope of the invention.In following embodiments, unless stated otherwise, agents useful for same, culture medium are
Commercial goods, method therefor are conventional method.1. culture medium
MRS+CaCO3Plate: MRS solid medium+10g/L CaCO3；
High sugar MRS+CaCO3Plate: MRS solid medium+10g/L CaCO3+ 180g/L glucose；
Screening and culturing medium: MRS fluid nutrient medium+10g/L CaCO3；
Acidogenic fermentation culture medium: glucose 80-160g/L, yeast extract 10g/L, sodium acetate 2g/L, KH2PO4 0.5g/
L、MgSO4·7H2O 0.5g/L、MnSO40.2g/L, Tween 80 1ml/L, CaCO3 40-80g/L。
2. high performance liquid chromatography detects the lactic acid in fermentation liquid
Chromatograph: 1260 Infinity II of Agilent Technologies；
Detector: RID；
Splitter: 300 × 7.8mm of Aminex HPX-87H Column；
Mobile phase: 0.05M sulfuric acid；
Flow: 0.5mL/min；
Sample volume: 20 μ L.
Lactic acid retention time is 14min or so.
3. biosensor detects the glucose and Pfansteihl in fermentation liquid
Instrument: SBA-40E type biosensor；
Enzyme membrane: D-Glucose enzyme membrane and Pfansteihl enzyme membrane；
Sample volume: 25 μ l.
4. the optical purity of high performance liquid chromatography detection lactic acid
Chromatograph: 1260 Infinity of Agilent Technologies；
Detector: wavelength 254nm, sensitivity 0.32AUFS；
Splitter: MCI GEL-CRS10W (3u) 4.6ID × 50mm；
Mobile phase: 0.002M copper sulphate；
Flow: 0.5mL/min；
Sample volume: 20 μ L.
D-ALPHA-Hydroxypropionic acid retention time is 9min or so, and Pfansteihl retention time is 11min or so, according to its calculated by peak area cream
The optical purity of acid.
Embodiment 1: strain isolation and identification
Strain isolation: the sterile saline of the yoghurt example from Xinjiang is diluted 1 times, mixes well 30min；It takes
It is coated on after stating 10 times of gradient dilutions of dilution containing 1% (w/v) CaCO3On the MRS solid medium of (neutralizer), 37 DEG C of cultures
24 hours, the biggish single colonie of picking transparent circle was inoculated with screening and culturing medium, and 37 DEG C are cultivated 24 hours, used high performance liquid chromatography detection
Total lactic acid content detects Pfansteihl yield with biosensor, selects lactic acid production height and is substantially not detectable Pfansteihl
Bacterial strain, i.e., the high lactic acid bacteria of D-ALPHA-Hydroxypropionic acid content in tunning.
Bacterial strain identification: the bacterial strain selected has that bacterium colony is smaller, round, milky, neat in edge are smooth, Gram's staining is in
Positive, microscopy cellular morphology is ellipse to rod-short, is mostly the features such as pairs of or bunchiness arrangement, substantially conforms to the bright beading of goldbeater's skin
The various features of bacterium.In turn, it confirmed that the bacterial strain and Leuconostoc mesenteroides are closest using 16S rDNA sequencing identification method, from
And it is accredited as Leuconostoc mesenteroides, it is named as Leuconostoc mesenteroides COLmD.
Specifically, by above-mentioned bacterial strains separation method isolated a large amount of production of lactic acid bacterial strains from yoghurt example, but
Wherein the overwhelming majority be produced with Pfansteihl based on (product D-ALPHA-Hydroxypropionic acid optical purity is less than 50%).In turn, in product D- cream
In a series of bacterial strains that sour optical purity is 80% or more, including 4 plants of Leuconostoc mesenteroides, wherein 3 plants of Leuconostoc mesenteroides
Product D-ALPHA-Hydroxypropionic acid optical purity is 85-90% or so, and the product D-ALPHA-Hydroxypropionic acid optical purity of Leuconostoc mesenteroides COLmD reaches
95% or so, this be also in the obtained all bacterial strains of screening product D-ALPHA-Hydroxypropionic acid optical purity it is highest.
Embodiment 2: strain mutagenesis
By the MRS that transfers after the activation of Leuconostoc mesenteroides COLmD bacterial strain obtained in embodiment 1 by way of plate streaking
Culture medium, 37 DEG C of 150rpm are cultivated to OD=1, are taken bacterium solution normal saline dilution to OD=0.6, are carried out atmospheric pressure at room plasma
Body (ARTP) mutagenesis.Using helium as the working gas of plasma, power 100W, gas flow 10slpm handle the time
120s, 150s, 180s are added 37 DEG C of 150rpm of MRS culture medium and recover 1 hour, carry out gradient to bacterium solution with sterile saline
It dilutes and is coated on high sugar MRS+CaCO3On solid plate, plate is placed in 37 DEG C of incubators and is cultivated, single colonie to be formed
And observe its molten calcium circle.
It selects bacterium colony in plate and grows the 37 DEG C of 150rpm trainings of the biggish single colonie screening and culturing medium of very fast and molten calcium circle
It supports for 24 hours, detects what screening glucose consumption rate was generated without Pfansteihl fastly and substantially with biosensor after culture
Bacterial strain.Fermentation shake flask experiment is carried out to the alternative bacterial strain filtered out, that is, by alternative strain inoculated MRS culture medium, 37 DEG C of 150rpm
It is incubated overnight acquisition seed liquor, is inoculated with above-mentioned seed liquor according to the ratio of 10% (v/v) in 30mL acidogenic fermentation culture medium, 37
DEG C 72 hours acquisition fermentation liquids of 150rpm shaking table culture.
After fermentation by the lactic acid in high performance liquid chromatography detection tunning, filter out and Leuconostoc mesenteroides
COLmD compares production of lactic acid fast speed and the higher bacterial strain of D-ALPHA-Hydroxypropionic acid optical purity.As a result, horizontal in 30mL shaking flask
Under, wild type Leuconostoc mesenteroides bacterial strain COLmD72h can fermentation production of D-lactic acid 53-55g/L, which is
About 95%；Leuconostoc mesenteroides bacterial strain 72h after the mutagenesis filtered out can fermentation production of D-lactic acid 65-69g/L, the D-ALPHA-Hydroxypropionic acid
Optical purity is greater than 99%.It is Leuconostoc mesenteroides COLmDH by the Leuconostoc mesenteroides Strain Designation after the mutagenesis filtered out
(also referred to as D-ALPHA-Hydroxypropionic acid produces bacterial strain COLmDH).The D-ALPHA-Hydroxypropionic acid produces bacterial strain COLmDH and is preserved in China on December 26th, 2017
Microbiological Culture Collection administration committee common micro-organisms center (CGMCC) (Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, in
Institute of microbiology, the academy of sciences, state), deposit number is CGMCC No.15131.
Embodiment 3: strain fermentation produces D-ALPHA-Hydroxypropionic acid
The production of D-ALPHA-Hydroxypropionic acid obtained in embodiment 2 bacterial strain COLmDH is inoculated with MRS culture medium, 37 DEG C of 150rpm are incubated overnight
Obtain seed liquor.Then, above-mentioned seed liquor is inoculated with according to the ratio of 10% (v/v) in 200mL acidogenic fermentation culture medium, 37 DEG C
72 hours acquisition fermentation liquids of 150rpm shaking table culture.Lactic acid production and optical voidness are measured by high performance liquid chromatography after fermentation
Degree.
As a result, wild-type strain COLmD 72h can fermentation production of D-lactic acid in the case where amplifying culture scale
56.2g/L, optical purity 97.7%；Bacterial strain COLmDH 72h after mutagenesis can fermentation production of D-lactic acid 68.4g/L (growth
21.7%), optical purity is 99.4% (increasing by 1.7%).In view of D-ALPHA-Hydroxypropionic acid industrial production usually requires that product optical purity
Reach 99% or more, the D-ALPHA-Hydroxypropionic acid production bacterial strain COLmDH after the mutagenesis provides the another selection that can meet industrial requirement.
Industrial applicibility
Above studies have shown that D-ALPHA-Hydroxypropionic acid production bacterial strain COLmDH of the invention is in D-ALPHA-Hydroxypropionic acid speed of production and product optical voidness
Degree aspect is significantly better than not modified Leuconostoc mesenteroides COLmD, and (speed of production improves 20% or more；Optical purity is increased to
99% or more).Therefore, that the present invention provides a kind of fermentation costs is low, environmental-friendly, D-ALPHA-Hydroxypropionic acid speed of production is fast and product light
The novel production bacterial strain of purity is high is learned, provides more preferably potential selection for the industrialization microbial fermentation production of D-ALPHA-Hydroxypropionic acid.
The preferred embodiment of the present invention has been described above in detail, still, during present invention is not limited to the embodiments described above
Detail a variety of modifications can be carried out to technical solution of the present invention, these changes within the scope of the technical concept of the present invention
Type all belongs to the scope of protection of the present invention.
Claims (10)
1. a kind of D-ALPHA-Hydroxypropionic acid produces bacterial strain COLmDH, the specific name of the D-ALPHA-Hydroxypropionic acid production bacterial strain COLmDH is the bright beading of goldbeater's skin
Bacterium (Leuconostoc mesenteroides) is preserved in Chinese microorganism strain preservation management committee on December 26th, 2017
Member can common micro-organisms center (CGMCC) (Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Chinese Academy of Sciences's microbe research
Institute), deposit number is CGMCC No.15131.
2. utilizing the method for D-ALPHA-Hydroxypropionic acid as described in claim 1 production bacterial strain COLmDH production D-ALPHA-Hydroxypropionic acid, which comprises
The D-ALPHA-Hydroxypropionic acid production bacterial strain COLmDH is cultivated in the medium, to generate and accumulate in the medium D- cream
Acid；And
D-ALPHA-Hydroxypropionic acid is collected from the culture medium.
3. method according to claim 2, wherein the described method comprises the following steps:
The D-ALPHA-Hydroxypropionic acid production bacterial strain COLmDH that freshly prepared D-ALPHA-Hydroxypropionic acid produces bacterial strain COLmDH or cryopreservation is inoculated in intestines
It is activated in film leukonid fluid nutrient medium；
It is incubated overnight obtained seed liquor；And
The seed liquor is seeded in D-ALPHA-Hydroxypropionic acid acidogenic fermentation culture medium to expand to cultivate, fermentation liquid is made.
4. method as claimed in claim 3, wherein the seed liquor is prepared by following process:
It picks from the plate D-ALPHA-Hydroxypropionic acid production bacterial strain COLmDH single colonie to be inoculated in seed liquid culture medium, with revolving speed 120-
180rpm cultivates 12 at 35-40 DEG C~for 24 hours to get seed liquor.
5. method as claimed in claim 4, wherein the seed liquor is prepared by following process:
It picks from the plate D-ALPHA-Hydroxypropionic acid production bacterial strain COLmDH single colonie to be inoculated in seed liquid culture medium, with revolving speed 120-
180rpm cultivates 12 at 37 DEG C~for 24 hours to get seed liquor.
6. method as described in claim 4 or 5, wherein the seed liquid culture medium is MRS fluid nutrient medium.
7. the method as described in any one of claim 3-6, wherein the fermentation liquid is prepared by following process:
Seed liquor is seeded in acidogenic fermentation culture medium with the inoculum concentration of 5%-10% volume, is existed with revolving speed 120-180rpm
72-96h continuously ferment at 35-40 DEG C to get fermentation liquid.
8. the method for claim 7, wherein the fermentation liquid is prepared by following process:
Seed liquor is seeded in acidogenic fermentation culture medium with the inoculum concentration of 5%-10% volume, with revolving speed 120-180rpm 37
72-96h continuously ferment at DEG C to get fermentation liquid.
9. method as claimed in claim 7 or 8, wherein the acidogenic fermentation culture medium include glucose, yeast extract,
Sodium acetate, phosphate, microelement and neutralizer.
10. method as claimed in claim 9, wherein the acidogenic fermentation culture medium includes following ingredient: glucose 80-
160g/L, yeast extract 10g/L, sodium acetate 2g/L, KH2PO4 0.5g/L、MgSO4·7H2O 0.5-1g/L、MnSO4 0.1-
0.2g/L, Tween 80 1ml/L and CaCO3 40-80g/L。
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| CN109628339A (en) * | 2018-12-18 | 2019-04-16 | 吉林中粮生化有限公司 | A kind of Pfansteihl production bacterial strain and the method using bacterial strain production Pfansteihl |
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| JP2009106278A (en) * | 2007-10-11 | 2009-05-21 | Daiso Co Ltd | Method for manufacturing d-lactic acid by fermentation |
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| CN109628339A (en) * | 2018-12-18 | 2019-04-16 | 吉林中粮生化有限公司 | A kind of Pfansteihl production bacterial strain and the method using bacterial strain production Pfansteihl |
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