CN106191140A - Fermentable produces strain HD 1025 and the method for glyceric acid - Google Patents
Fermentable produces strain HD 1025 and the method for glyceric acid Download PDFInfo
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Abstract
The present invention relates to a kind of microbial strains filtered out from rot fruitGluconobacter japonicas HD1025, its classification genus Acetobacteraceae (Acetobacteraceae) Bacterium gluconicum genus (Gluconobacter japonicus) this bacterial strain preserving number at China Committee for Culture Collection of Microorganisms's common micro-organisms center is: CGMCC No.12425.The present invention gives the method utilizing this bacterial strain to produce glyceric acid.This bacterial strain aerobic fermentation converts the glycerol of 50 200 g/L, the highest glyceric acid that can produce 52.39 g/L, great industrial production value.
Description
Technical field
The invention belongs to Biotechnology field, be specifically related to one aerobic microbiological fermentation glycerol and produce glyceric acid
Strain HD 1025 and utilize the method that this bacterial strain produces glyceric acid.
Background technology
Glyceric acid be the three carbon alkyd that formed of glycerol oxidation also known as 2,3-dihydroxypropionic acid, it is a kind of organic acid, its English
Literary fame is Glyceric acid, is abbreviated as GA, and molecular formula is C3H6O4, glyceric acid is present in the various plant of nature
In, and exist in equally in human body as a kind of intermediate metabolites.
According to the literature, glyceric acid and derivant thereof have the most important biological function.Such as: in human body, D-is sweet
Oleic acid can make stomach cell invigorate after doses ethanol stimulates thus promote ethanol decomposition metabolism (2007
Eriksson etc. confirm in mouse body) therefore it can be as the composition (patent that P Hai Nuo delivered in 2003 of antialcoholic drug
In elaborate the D-GLAC mechanism of action as antialcoholic drug).
The most also it has been reported that the glyceric acid in Canis familiaris L. body has the promotion anti-depressant function of hypocholesterolemic activity regulating liver-QI；Glycerol
The oligoesters that acid derives can show antitrypsic activity；Glyceric acid is better than it due to its biodegradability simultaneously
His some high polymers, therefore it may apply to the transport agent of medicine.In terms of food, glycerol has a lot of effect equally,
Such as: glyceric acid adds can make each component mix homogeneously, delicate mouthfeel lubricate, and can delay ice cream in ice cream
Melt.
The production method of glyceric acid has chemical method and bioanalysis two kinds.But, owing to bioanalysis report is less, existing market
The glyceric acid major part of upper sale is to be produced by chemical method.Comparing bioanalysis, chemical method has cost height, power consumption
Greatly, yield poorly and not there is the shortcomings such as stereo selectivity.Utilizing Production by Microorganism Fermentation glyceric acid, environment is gentle, yield
Height, method are easy and product has stereo selectivity, the most increasingly receive publicity.
Producing mainly by the microorganism containing alcoholdehydrogenase and aldehyde dehydrogenase with glycerol for substrate next life of glyceric acid
Produce.The bacterial strain part that can produce glyceric acid acid of report is as follows at present:G. thailandicus NBRC3172、G. Oxydans NBRC3292、G. Oxydans NBRC3292、G. frateurii NBRC3262、Gluconobactersp
NBRC3259、Acetobacter tropicalis NBRC16470、Gluconobacter frateurii NBRC103465
Deng.The report that external relevant microbial method produces glyceric acid is less, there is no any relevant glyceric acid at home and produces and produce
The report of glyceric acid bacterial strain screening.Therefore the industrialized production of glyceric acid is also faced with the most important difficulty, and one of them is exactly
The screening of superior strain.Therefore, filter out to autonomous innovation the microbial strains being resistant to high concentration glycerol, and work out
A kind of method that can produce high concentration glyceric acid is extremely urgent.
Summary of the invention
It is an object of the present invention to provide a strain be resistant to the glycerol of high concentration and can produce for raw material big with cheap glycerol
The new strains HD1025 of amount glyceric acid, this bacterial strain can be used for fermentable and produces glyceric acid.
The present invention gives the method utilizing this strain fermentation to produce glyceric acid, and the method is with glycerol as raw material.
A kind of fermentable produces the strain HD 1025 of glyceric acid, and its classification belongs to Acetobacteraceae
(Acetobacteraceae) Bacterium gluconicum genus (Gluconobacter japonicus) this bacterial strain is China Microbiological bacterium
The preserving number planting preservation administration committee common micro-organisms center is: CGMCC No.12425.Preservation date is 2016 05 month
09 day；Depositary institution address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3.Above-mentioned strain HD 1025 is from rotten fruit
Middle screening and go out, can the efficient oxidation glycerol production glyceric acid.
Use the method that above-mentioned bacterial strains HD1025 produces glyceric acid, its comprise the steps: by strain HD 1025 by 1～
The inoculum concentration of 10% accesses liquid fermentation medium, in 25～30 DEG C of fermentation culture 60～84h under aerobic condition；Described liquid
Fermentation medium consists of: 50～200 g/L glycerol, nitrogen source 6～20 g/L, 0.1～1 g/L KH2PO4, 0.1～1 g/L
K2HPO4, 1～5 g/L MgSO4·7H2O, 20～35 g/L CaCO3, surplus is water, and medium pH is 5～7.
Or, it is also possible to strain HD 1025 is first inoculated in primary-seed medium by the inoculum concentration of 1～10%, in 25
～30 DEG C of aerobic cultivation 6～24 h；Then by the inoculum concentration of 1～10%, cultured first order seed is accessed secondary seed to cultivate
In base, after 8～10 h, cultured secondary seed is accessed liquid fermentation medium again by the inoculum concentration of 1～10%；Wherein, institute
The composition stating primary-seed medium and secondary seed medium is: glucose 5 g/L, peptone 5 g/L, yeast powder 5 g/
L, MgSO4·7H2O 1 g/L, pH 6.5, surplus is water.
Concrete, described glycerol is food grade glycerin or pharmaceutical grade glycerol.
Concrete, described nitrogen source be yeast powder, Carnis Bovis seu Bubali cream, peptone, Semen Maydis pulp, carbamide and inorganic ammonium salt (as ammonium sulfate,
Ammonium nitrate or ammonium chloride etc.) in one or more mixture.
In the present invention, set up the model of a kind of rapid screening high yield glyceric acid bacterial strain, there is alcoholdehydrogenase and aldehyde dehydrogenation
The microorganism of enzyme can produce organic acid in the screening culture medium with glycerol as sole carbon source, and this organic acid is so that screen
The pH of culture medium local changes, hence in so that the acid-base indicator color in screening culture medium changes, the most permissible
The bacterial strain of organic acid is produced in the screening of rapid preliminary, the most again by this inoculation in fermentation medium, culture medium a period of time
After utilize high performance liquid chromatograph (HPLC) that fermentation liquid carries out qualitative and detection by quantitative, and then sift out the bacterium of high yield glyceric acid again
Strain.
The samples such as the soil that the present invention enriches from rotten fruit, vegetable, sewage and organic matter sample, prepares bacterium solution and be coated with
It is distributed in screening culture medium cultivation.Then the inoculation that picking surrounding media color changes in fermentation medium,
Cultivate 2-3 d for 30 DEG C, take fermentation liquid and be centrifuged, dilute 100 times, utilize HPLC qualitative and quantitative detection.Big through to multiple samples
Amount bacterial strain screening work, the most just filters out tens strains from the near hundreds of strain antibacterials separated and produces the bacterial strain of glyceric acid, finally
Multiple sieve obtains the product glyceric acid HD1025 bacterial strain that hereditary stability is good.What present invention screening obtained can effectively glycerine converting produce sweet
The microorganism HD1025 of oleic acid, the Physiology and biochemistry qualification identified through 16S rRNA and carry out according to primary Jie Shi Bacteria Identification handbook,
Think this bacterial strain belong to acetic acid Cordycepps (Acetobacteraceae) Gluconobacter (Gluconobacter)Gluconobacter japonicus。
The outstanding advantages of the inventive method is to utilize cheap glycerol to carry out fermenting and producing high value added product glycerol for raw material
Acid, the sustainable production of the method, and environmentally friendly.
Accompanying drawing explanation
Fig. 1 isGluconobacter japonicus The flat-plate bacterial colony photo of HD1025；
Fig. 2 isGluconobacter japonicus The stereoscan photograph of HD1025；
Fig. 3 isGluconobacter japonicus The 16S rDNA complete sequence sequencing result of HD1025；
Fig. 4 is the cladogram built based on BLAST result.
Detailed description of the invention
Below in conjunction with embodiment, technical scheme is discussed in detail further, but protection scope of the present invention
It is not limited thereto.
Following presentGluconobacter japonicus The screening of HD1025 bacterial strain, qualification and fermenting and producing glycerol
The detailed process of acid.
(1) screening of bacterial strain:
1, primary dcreening operation:
Different location sampling in urban district, Kaifeng, sampling kind includes the soil that rotten fruit, vegetable, sewage and organic matter are abundant
Earth etc., altogether sampling 30 parts.The sample of acquirement is carried out grinding process, above-mentioned sample is added in sterilized water, is placed in constant-temperature table
In carry out 2 h that vibrate, then take supernatant bacterium solution and carry out gradient dilution, take 10-4、10-5、10-6The bacterium solution 200 μ L of three gradients is coated with
It is distributed in screening culture medium (glycerol 150 g/L, peptone 9 g/L, yeast powder 1 g/L, MgSO4·7H2O 1 g/L, bromocresol purple
0.02 g/L, agar powder 20 g/L；PH 6.5) in, in 30 DEG C of constant incubators, cultivate 2-4 d.Picking colony form differs
Single bacterium colony that relatively big and this periphery of bacterial colonies culture medium color changes sieves the most again.
Multiple sieve:
Over one hundred strain bacterial strain of above-mentioned primary dcreening operation gained is inoculated in batches fermentation medium (glycerol 100 g/L, peptone 9 g/L,
Yeast powder 1 g/L, KH2PO40.9 g/L, K2HPO40.1 g/L, MgSO4·7H2O 1 g/L, CaCO320 g/L, surplus is
Water, pH 6.5) in, in 30 DEG C of constant-temperature tables, 220 rpm cultivate 3 d.Take fermentation liquid 1 mL under 8000 r/min, be centrifuged 5
Min, takes supernatant 0.5 mL and dilutes 100 times, utilizes the content of glyceric acid in high performance liquid chromatograph (HPLC) detection fermentation liquid.
Chromatographic condition is: flowing phase: containing 5 mM H of 20% acetonitrile2SO4；Flow velocity: 0.3 mL/min；Column temperature: 60 DEG C；Chromatographic column:
Aminex HPX-87H ion chromatographic column (7.8 mm × 300 mm, 9 μm)；Detector: RI detector, UV detector (detection ripple
Long 210 nm).
The yield of wherein most bacterial strain is the lowest, and the yield of the most a few strain bacterial strains is higher.By the above-mentioned bacterial strain sieving gained again
Carry out inclined-plane preservation, be subsequently placed in 4 DEG C of refrigerators preservation.The bacterial strain that following table lists a few strain yield higher the results are shown in Table 1.
The product glyceric acid situation of table 1 part test bacterial strain
。
(2)Gluconobacter japonicusThe qualification of bacterial strain:
What screening obtained can effectively produce glyceric acid and production effect significant microbial strains HD1025, and the cell of this bacterium is short
Shaft-like it is close to round, does not moves, without spore；Belonging to Gram-negative, bacterium colony is trained under the conditions of 30 DEG C on the flat board containing glycerol
Support that bacterium colony after 4 d is less, moistening, circular, protuberance, edge waveform, opaque, positive and negative solid colour are pale, and diameter can
Reach 1 ~ 3 mm(and see Fig. 1 and Fig. 2).
By " primary Jie Shi Bacteria Identification handbook ", " common bacteria classification manual ", " Bacterium gluconicum belongs to and classifying and main
Application progress " (Feng waits quietly, JOURNAL OF MICROBIOLOGY, 2010,30(2): 86-90) and "Gluconobacter japonicus sp. nov., an acetic acid bacterium in the Alphaproteobacteria》
(International Journal of Systematic and Evolutionary Microbiology 2009,59:
466 471) physio-biochemical characteristics qualification (the results are shown in Table 2) is carried out.
According to Gram’s staining, catalase, oxidase, reduction nitrate, liquefaction gelatin, produce indole, Oxidation of Alcohol to second
Acid, acetic oxide salt to CO2And H2The raw ketone of O, many alcohols, hydrolysis starch, D-xylose produce acid, D-Glucose produces acid etc.GluconobacterThe characteristic physiological biochemical reaction result belonged to identifies that this bacterial strain belongs toGluconobacterBelong to.According to forming 5-
Ketone group gluconic acid, formation glyceric acid, the upper chromogenic element of GYC, maltose produce acid, the growth of D-R alcohol, erythritol growth
Identify that this bacterial strain belongs to Deng physio-biochemical characteristics between plantingGluconobacter japonicus, intend namedGluconobacter japonicus HD1025。
Meanwhile, Dalian treasured biological engineering company limited is entrusted HD1025 bacterial strain to carry out the order-checking of 16S rDNA complete sequence (altogether
1398bp, result is shown in Fig. 3).By the 16S rDNA gene order of related strain in BLAST retrieval GenBank on NCBI website
(see Table 3), by MEGA 4.0 software building phylogenetic analysis (see figure 4).Based on 16S rDNA sequence analysis and structure cladogram
Analysis draw HD1025 withGluconobacter japonicus、Gluconobacter frateuriiSibship is relatively near,
But withGluconobacter frateuriiDifference somewhat is there is in bacterial strain on bio-chemical characteristics.Strain HD 1025 can
To be defined asGluconobacterBelong to。
From above-mentioned qualification result, strain HD 1025 belong to acetic acid Cordycepps (Acetobacteraceae) Bacterium gluconicum
Belong to (Gluconobacter japonicus).
Table 2 physio-biochemical characteristics qualification result synopsis
BLAST Yu HD1025 bacterial strain 16S rDNA similarity partial results on table 3 NCBI
。
(3) bacterial strain is utilizedGluconobacter japonicus HD1025 fermenting and producing glyceric acid:
Embodiment 1
Utilize the method that described strain HD 1025 fermentation glycerol produces glyceric acid, particularly as follows: by strain HD 1025 first by 10% connect
The amount of kind is inoculated in primary-seed medium, and aerobic cultivation 24 h in 30 DEG C of constant-temperature tables, the inoculum concentration by 10% is by cultivation well
First order seed access in secondary seed medium, aerobic cultivation 10 h in 30 DEG C of constant-temperature tables, then by cultured two
Level seed accesses in the liquid fermentation medium of the glycerol containing variable concentrations by the inoculum concentration of 10%, the culture medium that will have inoculated
It is placed in 30 DEG C of constant incubators aerobic cultivation 2-3 d under 220 rpm.HPLC is utilized to examine after taking the centrifugal dilution of fermentation liquid
Survey.Record result as shown in table 4.
Primary-seed medium: glucose 5 g/L, peptone 5 g/L, yeast powder 5 g/L, MgSO4·7H2O 1 g/L,
PH 6.5, surplus is water.
Secondary seed medium: glucose 5 g/L, peptone 5 g/L, yeast powder 5 g/L, MgSO4·7H2O 1 g/L,
PH 6.5, surplus is water.
One-level, secondary seed medium sterilising temp and time are respectively as follows: 115 DEG C, 30 min.
Liquid fermentation medium: glycerol 50～250 g/L, peptone 9 g/L, yeast powder 1 g/L, KH2PO4 0.5 g/
L, K2HPO40.1 g/L, MgSO4·7H2O 1 g/L, CaCO325 g/L, surplus is water, pH 6.5.Glycerol is that food stage is sweet
Oil.
The sterilising temp of liquid fermentation medium and time are respectively as follows: 121 DEG C, 30 min.CaCO3 160 DEG C of dry heat sterilizations 1
h。
The glyceric acid yield of strain HD 1025 under the different glycerol concentration of table 4
。
Embodiment 2:
Utilize the method that described strain HD 1025 fermentation glycerol produces glyceric acid, particularly as follows: strain HD 1025 to be pressed the inoculation of 5%
Amount be directly accessed liquid fermentation medium (described liquid fermentation medium consists of: glycerol 100 g/L, peptone 9.69 g/L,
Yeast powder 5 g/L, KH2PO4 0.9 g/L, K2HPO40.3 g/L, MgSO4·7H2O 2 g/L, CaCO320 g/L, culture medium
PH 6.0), in 30 DEG C of fermentation culture 72 h under aerobic condition；Recording the content of glyceric acid in fermentation liquid is 45.78 g/L.
Embodiment 3:
Utilize the method that described strain HD 1025 fermentation glycerol produces glyceric acid, particularly as follows: strain HD 1025 to be pressed the inoculation of 10%
Amount is directly accessed liquid fermentation medium, and (described liquid fermentation medium consists of: glycerol 60 g/L, Semen Maydis pulp 9 g/L, yeast
Powder 1g/L, KH2PO4 0.5 g/L, K2HPO40.5 g/L, MgSO4·7H2O 2 g/L, CaCO320 g/L, medium pH
6.5), in 30 DEG C of fermentation culture 60 h under aerobic condition；Recording the content of glyceric acid in fermentation liquid is 15.5 g/L.
Embodiment 4:
Utilize the method that described strain HD 1025 fermentation glycerol produces glyceric acid, particularly as follows: strain HD 1025 to be pressed the inoculation of 10%
Amount is directly accessed liquid fermentation medium, and (described liquid fermentation medium consists of: glycerol 150 g/L, Carnis Bovis seu Bubali cream 9 g/L, ferment
Female powder 3 g/L, KH2PO4 0.9 g/L, K2HPO40.1 g/L, MgSO4·7H2O 3 g/L, CaCO330 g/L, culture medium
PH 6.5), in 30 DEG C of fermentation culture 72 h under aerobic condition；Recording the content of glyceric acid in fermentation liquid is 54.59 g/L.
Embodiment 5:
Utilize the method that described strain HD 1025 fermentation glycerol produces glyceric acid, particularly as follows: strain HD 1025 to be pressed the inoculation of 10%
Amount is directly accessed liquid fermentation medium, and (described liquid fermentation medium consists of: glycerol 200 g/L, peptone 10 g/L, urine
Element 5g/L, KH2PO4 0.2 g/L, K2HPO40.9 g/L, MgSO4·7H2O 5 g/L, CaCO335 g/L, medium pH
6.5), in 30 DEG C under aerobic condition, 240 rpm fermentation culture 84 h；Recording the content of glyceric acid in fermentation liquid is 56.33
g/L。
Embodiment 6:
Utilize the method that described strain HD 1025 fermentation glycerol produces glyceric acid, particularly as follows: strain HD 1025 to be pressed the inoculation of 5%
Amount be directly accessed liquid fermentation medium (described liquid fermentation medium consists of: glycerol 150 g/L, ammonium sulfate 9.69 g/L,
Yeast powder 10 g/L, KH2PO4 0.9 g/L, K2HPO40.1 g/L, MgSO4·7H2O 1 g/L, CaCO325 g/L, culture medium
PH 6.5), in 30 DEG C of fermentation culture 72 h under aerobic condition；Recording the content of glyceric acid in fermentation liquid is 43.60 g/L.
Claims (5)
1. the strain HD 1025 of a fermentable production glyceric acid, its classification belongs to Acetobacteraceae
(Acetobacteraceae) Bacterium gluconicum genus (Gluconobacter japonicus) this bacterial strain is China Microbiological bacterium
The preserving number planting preservation administration committee common micro-organisms center is: CGMCC No.12425.
2. use the method that strain HD 1025 described in claim 1 produces glyceric acid, it is characterised in that comprise the steps: by
Strain HD 1025 accesses liquid fermentation medium, in 25～30 DEG C of fermentation culture 60 under aerobic condition by the inoculum concentration of 1～10%
～84h；Described liquid fermentation medium consists of: 50～200 g/L glycerol, nitrogen source 6～20 g/L, 0.1～1 g/L
KH2PO4, 0.1～1 g/L K2HPO4, 1～5 g/L MgSO4·7H2O, 20～35 g/L CaCO3, surplus is water, culture medium
PH is 5～7.
The method producing glyceric acid the most according to claim 2, it is characterised in that by strain HD 1025 first by 1～10% connect
The amount of kind is inoculated in primary-seed medium, in 25～30 DEG C of aerobic cultivation 6～24 h；Then cultured first order seed is pressed
1～10% inoculum concentration access in secondary seed medium, by cultured secondary seed by 1～the inoculation of 10% after 8～10 h
Amount accesses liquid fermentation medium again；Wherein, the composition of described primary-seed medium and secondary seed medium is: Fructus Vitis viniferae
Sugar 5 g/L, peptone 5 g/L, yeast powder 5 g/L, MgSO4·7H2O 1 g/L, pH 6.5, surplus is water.
The method producing glyceric acid the most according to claim 2, it is characterised in that described glycerol is food grade glycerin or doctor
Medicine level glycerol.
The method producing glyceric acid the most according to claim 2, it is characterised in that described nitrogen source is yeast powder, Carnis Bovis seu Bubali cream, egg
One or more mixture in white peptone, Semen Maydis pulp, carbamide and inorganic ammonium salt.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109207530A (en) * | 2018-11-14 | 2019-01-15 | 河南大学 | A method of dihydroxyacetone (DHA) is produced with Japanese gluconobacter suboxydans strain HD 1025 |
| CN112063546A (en) * | 2020-08-11 | 2020-12-11 | 中国科学院青岛生物能源与过程研究所 | Gluconobacter CDT2 and screening method and application thereof |
| CN114196790A (en) * | 2021-12-24 | 2022-03-18 | 内蒙古金达威药业有限公司 | Production control method of glyceric acid |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109207530A (en) * | 2018-11-14 | 2019-01-15 | 河南大学 | A method of dihydroxyacetone (DHA) is produced with Japanese gluconobacter suboxydans strain HD 1025 |
| CN112063546A (en) * | 2020-08-11 | 2020-12-11 | 中国科学院青岛生物能源与过程研究所 | Gluconobacter CDT2 and screening method and application thereof |
| CN112063546B (en) * | 2020-08-11 | 2023-02-28 | 中国科学院青岛生物能源与过程研究所 | Gluconobacter CDT2 and screening method and application thereof |
| CN114196790A (en) * | 2021-12-24 | 2022-03-18 | 内蒙古金达威药业有限公司 | Production control method of glyceric acid |
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