CN102391976A - Strain HD924 for producing dihydroxyacetone by microbial fermentation and method - Google Patents
Strain HD924 for producing dihydroxyacetone by microbial fermentation and method Download PDFInfo
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Abstract
The invention relates to a strain HD924 for producing dihydroxyacetone by microbial fermentation. The strain HD924 is classified into Acetobacteraceae Gluconobacter Gluconobacterfrateurii, and its collection number in the common micro-organism center of the CCCCM is CGMCC No.5397. The invention also provides a method for producing dihydroxyacetone by using the strain to ferment glycerin. dihydroxyacetone can be remarkably accumulated under aerobic conditions with the maximum of 170g/L. By the adoption of the method provided by the invention, industrial production can be realized.
Description
Technical field
The invention belongs to biological manufacturing field, be specifically related to produce the strain HD 924 of otan with a kind of aerobic microbiological ferment glycerin
WithUtilize this strain fermentation glycerine to produce the method for otan.
Background technology
Otan claims 1 again, the 3-otan, and English is called 1, and 3-dihydroxyacetone or dihydroxyacetone are abbreviated as DHA, and molecular formula is C
3H
6O
3, molecular weight is 90.08, is the simplest three carbon ketoses, outward appearance is white or the crystallization of off-white powder shape, has sweet, cool taste, is prone to moisture absorption and decomposition.Be that (1, crystallization 4-Dioxane) becomes monomer to dimer after dissolving or heating under the general state.
Otan is a kind of natural existence, the simplest water miscible ketose, has biodegradability, edible and to human body and environment toxicological harmless; Have several active groups in its molecule, but the multiple reaction of catalysis.Its verivate is very important one type of midbody during organic chemistry synthesizes.Therefore obtained practical application widely abroad as a kind of important chemical material, medicine intermediate and functional additive.Because contain three kinds of functional groups in the otan molecule, chemical property is active, and wide participation is a kind of important chemosynthesis midbody such as chemical reactions such as polymerization, condensations.In industry, DHA can effectively reduce divinyl-vinylbenzene mixture; Effectively catalysis formaldehyde is to the condensation reaction of aldol, hydroxyl ketone; But synthesizing heterocyclic compounds,, triglyceride level, ketone position substitution compound; The biomaterial product that also can be used for synthetic non-toxic degradable.In addition, otan also can be used as a kind of good active coloring ingredient and is widely applied in the makeup.One of maximum characteristics of DHA are exactly to have extremely strong sun-proof property, and can be combined in skin surface with the skin surface free amino group and form a fine and closely woven film, reflected sunlight, shielding ultraviolet rays stops the moisture of skin excessive vaporization, plays and well preserves moisture and sunscreen effect.At present, DHA has been applied in the treatment of hypoglycemia and treatment of diabetes and some viral dermatosis (as hickie and vitiligo are just had significant curative effect) as medicine.As medicine intermediate, DHA can synthesize the medicine of some treatment cardiovascular disordeies.It also can be directly as a kind of Anti-virus agent simultaneously, and as in the egg embryo culture, use DHA can suppress the infection of ewcastle disease virus greatly, kills 51 ~ 100% ewcastle disease virus.Synthesize corresponding verivate with DHA as midbody in addition, can have the effect of AIDS virus resisting.Aspect agricultural and food, otan can be used as fodder additives, in pig feed, adds the mixture of a certain amount of otan and pyruvate salt, and the lipid content that can reduce pork increases lean ratio.In meals, add otan and can improve aerobic endurance, also can change the aspects such as variation of organism metabolism rate and substrate utilization blood fat.
The suitability for industrialized production of otan mainly is to utilize that to contain the glycerol dehydrogenase mikrobe be fermenting substrate production with glycerine.At present the maximum mikrobe that can produce glycerol dehydrogenase of report be gluconobacter suboxydans belong to (
Gluconobacter) and genus acetobacter (
Acetobacter), the major microorganisms resource that can produce otan is following:
Bacillus xylinum, Acinetobacter sp. strainJC1 DSM 3803
, Acetobacter xylinumA-9
, Gluconobacter melanogenusIFO 3293
, Gluconobacter melanogenusIFO 3294
, Hansenula polymorphaCBS 4732
, Candida boidinii2201
, Klebsiella aerogenes, Escherichia coli(strain ECFS)
, Gluconobacter oxydansATCC 621
, Pichia membranifaciens, Bacillus licheniformisB-05571
, AcetomonasSp etc.Less about the Microbial resources report of high yield otan both at home and abroad.Therefore, filter out to autonomous innovation the microorganism strains that can tolerate high density glycerine, and it is extremely urgent to work out a kind of method that can production high density otan.
Summary of the invention
The object of the invention is to provide a kind of has the strain HD 924 of certain tolerance to high density glycerine, and this bacterial strain can be used for microbial fermentation and produces otan.The present invention gives the method for utilizing these strain HD 924 fermentative prodn otans, and this method is a raw material with the glycerine of cheapness, and glycerol conversion yield is higher.
For realizing above-mentioned purpose, the present invention adopts following technical scheme:
A kind of microbial fermentation is produced the strain HD 924 of otan, its minute the generic Acetobacteraceae (
Acetobacteraceae) the gluconobacter suboxydans genus (
Gluconobacter) not the Tuo Shi gluconobacter suboxydans (
Gluconobacter frateurii), this bacterial strain at the preserving number at China Committee for Culture Collection of Microorganisms common micro-organisms center is: CGMCC No. 5397, preservation date on October 28th, 2011, depositary institution address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City.
Utilize said strain HD 924 ferment glycerins to produce the method for otan, be specially: strain HD 924 is inserted fermention medium by 1～10% inoculum size, under aerobic condition in 28～30 ℃ of fermentation culture 48～72h; Said fermention medium consists of: glycerine 50～350g/L, nitrogenous source 5～20g/L, lime carbonate 0.1～15g/L, water preparation, medium pH 4～7.
Preferably, also can strain HD 924 first inoculum sizes by 1～10% be inoculated in the first order seed substratum, in 28～30 ℃ of aerobic cultivation 4～24 h; Then cultured first order seed is inserted in the secondary seed medium by 1～10% inoculum size, behind 8～10 h cultured secondary seed is inserted fermention medium again by 1～10% inoculum size; Wherein, consisting of of said first order seed substratum: glycerine 30～50g/L, yeast powder 5～15g/L, KH
2PO
40.1～0.5g/L, water preparation, pH nature; Consisting of of said secondary seed medium: glycerine 30～50g/L, yeast powder 5～15g/L, CaCO
30.1～0.5g/L, the water preparation, pH 4.0～7.0.
Concrete, said glycerine is preferably food grade glycerine, technical grade glycerine or saponification raw glycerine etc.Said nitrogenous source is selected from yeast powder, Carnis Bovis seu Bubali cream, peptone, steeping water, urea or inorganic ammonium salt etc.
Can regulate the pH value of substratum through the ordinary method of this area, as adding mineral acid alkali salts such as hydrochloric acid, sodium hydroxide, yellow soda ash.Wherein, the sterilising conditions of said fermention medium, first order seed substratum and secondary seed medium is 115～121 ℃, 15～25min.
In the present invention, set up the model of a screening high yield otan.Can utilize glycerine as carbon source for growth owing to have the mikrobe of glycerol dehydrogenase, and produce otan, therefore designing with glycerine is the selectivity flat board of sole carbon source, filters out the bacterial strain that can utilize glycerine.On flat board, choosing bigger bacterium colony to shake flask fermentation screens.Characteristics of the present invention be from rotten fruit, separate the not Tuo Shi gluconobacter suboxydans obtain effectively producing otan (
Gluconobacter frateurii) HD924, it can accumulate otan (40-170 g/L) significantly under aerobic condition, be up to 170 g/L, can realize suitability for industrialized production.The outstanding advantage of the inventive method is to utilize cheap glycerine to come the fermentative prodn otan for raw material, this method Sustainable Production, and environmentally friendly.
Description of drawings
Fig. 1 is the flat-plate bacterial colony photo (* 10) of not Tuo Shi gluconobacter suboxydans HD924;
Fig. 2 is the stereoscan photograph (* 10000) of not Tuo Shi gluconobacter suboxydans HD924;
Fig. 3 is the 16S rDNA complete sequence sequencing result of not Tuo Shi gluconobacter suboxydans HD924;
Fig. 4 is the evolutionary tree that makes up based on BLAST result.
Embodiment
(1) not the Tuo Shi gluconobacter suboxydans (
Gluconobacter frateurii) screening of HD924:
1, primary dcreening operation:
From rotten fruit, take a sample, after the pulverizing, get 10g and add the 90mL SPSS, 1 h that vibrates gets supernatant, is diluted to a series of concentration gradients.Get 10
-5, 10
-6, 10
-7Three gradient 0.2 mL coat plate culture medium, and (plate culture medium consists of: glycerine 30g/L, yeast powder 15g/L, agar powder 15g/L, KH
2PO
43g/L, the pH nature), cultivate 3 ~ 4 d, obtain single bacterium colony for 28 ℃.It is dull and stereotyped uniformly to choose bacterium colony, and picking 10-20 is seeded to fermention medium respectively than macrocolony (every strain bacterial strain is done one and shaken bottle, and fermention medium consists of: glycerine 100 g/L, yeast powder 10g/L, KH from it
2PO
43g/L, the pH nature), 28 ℃ of fermentation culture 48h.
Get 12000 rev/mins of centrifugal 5 min of 1.5mL fermented liquid then; Get 0.5 mL supernatant; (diphenylamine solution is prepared as follows: with 0.6g pentanoic and 54 mL anhydrous acetic acid mixings to add the 4.5mL diphenylamine solution; Slowly add the 6mL vitriol oil again, mixing promptly gets), measure OD behind the boiling water bath 15min
608, and calculate according to this otan content (concrete measuring method can be referring to Liu Yupeng, Li Hua etc., in the fermented liquid 1, the spectrophotometry of 3-otan, Chinese Journal of Pharmaceuticals, 2011,42 (11), 834-837).
Choose and produce the higher over one hundred strain bacterial strain of otan, (slant medium consists of: glycerine 30g/L, yeast powder 15g/L, agar powder 15g/L, KH under 4 ℃ of conditions, to preserve its respective ramp
2PO
43g/L, the pH nature, glycerine is food grade).
2, multiple sieve:
The bacterial strain that primary dcreening operation is kept transfer in the triangular flask that the 30mL fermention medium is housed (every strain bacterial strain is done three and is shaken the parallel appearance of bottle, fermention medium form with primary dcreening operation in fermention medium identical), 200 rev/mins, 28 ℃ of aerobic cultivation 48h.
Get 12000 rev/mins of centrifugal 5min of 1.5mL fermented liquid, get supernatant, otan content is measured with the HPLC method in the dilution back.The HPLC condition is following: chromatographic column Aminex HPX-87H post (7.8 mm * 300 mm, 9 μ m); Moving phase: 8 mmol/L sulfuric acid; Detector: RI detector, UV detector (detecting wavelength 215 nm); 50 ℃ of column temperatures; Flow velocity 0.5 ml/min; Sample size 20 μ l.
Most bacterial strain otan content are lower, and there only have 3-8-4 bacterial strain (this strain number of laboratory is HD924) to produce otan to be remarkable, is 45.7 g/L.The tens of preferably surplus strain bacterial strains of product otan effect have been provided in the table 1.
(2) not the Tuo Shi gluconobacter suboxydans (
Gluconobacter frateurii) evaluation of HD924:
What screening obtained can effectively accumulate otan and obvious results microorganism strains HD924: the cell of this bacterium is rod-short, does not move, no gemma.Gram-negative, bacterium colony bacterium colony after cultivating 3～4d under 28 ℃ of conditions on the glycerine flat board is less, moistening, circular, protuberance, neat in edge, translucent, be faint yellow, diameter can reach 1.6-2.5 mm (seeing Fig. 1 and Fig. 2).
By " uncle Jie Shi Bacteria Identification handbook (the 8th edition) ", " gluconobacter suboxydans belongs to classification and the main progress of using thereof " (Feng waits quietly, JOURNAL OF MICROBIOLOGY, 2010,30 (2): 86-90) with "
Gluconobacter sphaericus(Ameyama 1975) comb. nov, a brown pigment-producing acetic acid bacterium in the
Alphaproteobacteria" (Taweesak Malimas et al.,
J Gen Appl Microbiol, 2008,54:211-220) carry out physio-biochemical characteristics and identify (result sees table 2).
According to gramstaining, catalase, oxydase, reduction nitrate salt, liquefy gelatin, product indoles, oxidation ethanol to acetate, oxidation ethanol to acetate, acetic oxide salt to CO
2And H
2O, many alcohols are given birth to ketone, hydrolyzed starch, the acid of D-wood sugar product, the acid of D-glucose product, otan etc.
GluconobacterThe characteristic biochemical reactions result who belongs to identifies that this bacterial strain belongs to
GluconobacterBelong to.This bacterial strain of physio-biochemical characteristics evaluation belongs between kind such as grow when forming 5-ketone group glucono-, last products of GYC pigment, the acid of SANMALT-S product, the growth of D-arabitol, erythritol, no nicotinic acid
Gluconobacter frateurii, intend called after
Gluconobacter frateuriiHD924.
Simultaneously, entrust Dalian precious biotechnology ltd that the HD924 bacterial strain is carried out 16S rDNA complete sequence order-checking (be total to 1364bp, the result sees Fig. 3).On the NCBI website,, analyze (see figure 4) with MEGA 4.0 software building evolutionary trees with the 16S rDNA gene order (seeing table 3) of relevant bacterial strain among the BLAST retrieval GenBank.Based on 16S rDNA sequential analysis with make up the evolutionary tree analysis draw HD924 with
Gluconobacter frateurii,
Gluconobacter oxydans, Gluconobacter thailandicusSibship is nearer, and strain HD 924 can be confirmed as
GluconobacterBelong to
Can know by above-mentioned qualification result, strain HD 924 genus acetic acid Cordycepps (
Acetobacteraceae) the gluconobacter suboxydans genus (
Gluconobacter) not the Tuo Shi gluconobacter suboxydans (
Gluconobacter frateurii), this bacterial strain at the preserving number at China Committee for Culture Collection of Microorganisms common micro-organisms center is: CGMCC No. 5397.
(3) utilize strain HD 924 fermentative prodn otans:
Utilize said strain HD 924 ferment glycerins to produce the method for otan, be specially: strain HD 924 first inoculum sizes by 10% are inoculated into (consisting of of said first order seed substratum: glycerine 30g/L, yeast powder 15g/L, KH in the first order seed substratum
2PO
40.3g/L, the pH nature), in 28 ℃ of aerobic cultivation 24h; Then cultured first order seed is inserted (consisting of of said secondary seed medium: glycerine 30g/L, yeast powder 15g/L, CaCO in the secondary seed medium by 5% inoculum size
30.3g/L pH 6.0), behind 8 h cultured secondary seed is inserted (250mL triangular flask in the fermention medium that contains different glycerol concentrations respectively by 5% inoculum size; Liquid amount 50mL) rotating speed is 200 rev/mins; 28 ℃ of condition bottom fermentation 48 h measure otan content, and experimental result is seen table 4.
Wherein, said fermention medium consists of: glycerine 50 ~ 350g/L, yeast powder 10g/L, CaCO
33g/L, pH 6.0, and glycerine is food grade.(when adopting triangular flask to cultivate, 121 ℃ of seed sterilising temps, 15 min; 115 ℃ of fermention medium sterilising temps, 25 min, CaCO
3160 ℃ of dry sterilization 1 h; Down together).
Embodiment 2
Cultured secondary seed is inserted (250mL triangular flask in the fermention medium that glycerine (glycerine is respectively from food grade glycerine, technical grade glycerine and saponification raw glycerine) concentration is 100g/L respectively by 5% inoculum size; Liquid amount 50mL) rotating speed is 200 rev/mins; 28 ℃ of condition bottom fermentation 72h; Measure otan content, experimental result is seen table 5.Wherein, said fermention medium consists of: glycerine 100g/L, yeast powder 10g/L, CaCO
33g/L, pH 6.0; Food grade glycerine purity is 99.5%, and technical grade glycerine purity is 95%, and the saponification raw glycerine is the useless glycerine (purity 60%) of soapmaking industry.The cultivation of I and II seed is identical with embodiment 1.
Embodiment 3
Utilize said strain HD 924 ferment glycerins to produce the method for otan, be specially: strain HD 924 is directly inserted fermention medium (250mL triangular flask, liquid amount 50mL by 3% inoculum size; Said fermention medium consists of: food grade glycerine 100g/L, peptone 8g/L, lime carbonate 5g/L, medium pH 7) in, 200 rev/mins of rotating speeds, under aerobic condition in 30 ℃ of fermentation culture 72 h; Record that otan content is 67.4 g/L in the fermented liquid.
Embodiment 4
Utilize said strain HD 924 ferment glycerins to produce the method for otan, be specially: strain HD 924 is directly inserted fermention medium (250mL triangular flask, liquid amount 50mL by 8% inoculum size; Said fermention medium consists of: food grade glycerine 100g/L, steeping water 20g/L, lime carbonate 10g/L, medium pH 5) in, 200 rev/mins of rotating speeds, under aerobic condition in 30 ℃ of fermentation culture 72 h; Record that otan content is 73.9 g/L in the fermented liquid.
Embodiment 5
Utilize said strain HD 924 ferment glycerins to produce the method for otan, be specially: strain HD 924 is directly inserted fermention medium (250mL triangular flask, liquid amount 50mL by 2% inoculum size; Said fermention medium consists of: food grade glycerine 100g/L, urea 8g/L, lime carbonate 12g/L, medium pH 4) in, 200 rev/mins of rotating speeds, under aerobic condition in 28 ℃ of fermentation culture 48 h; Record that otan content is 10.5 g/L in the fermented liquid.
Embodiment 6
Utilize said strain HD 924 ferment glycerins to produce the method for otan, be specially: cultured secondary seed is directly inserted fermention medium (250 mL triangular flasks, liquid amount 50 mL by 10% inoculum size; Said fermention medium consists of: food grade glycerine 100g/L, Carnis Bovis seu Bubali cream 10 g/L, lime carbonate 15g/L, medium pH 7) in, 200 rev/mins of rotating speeds, under aerobic condition in 28 ℃ of fermentation culture 48 h; Record that otan content is 69.9 g/L in the fermented liquid.The cultivation of I and II seed is identical with embodiment 1.
Embodiment 7
Utilize said strain HD 924 ferment glycerins to produce the method for otan, be specially: it is that (fermentative medium formula is: glycerine 160g/L, yeast powder 15g/L, CaCO for the 7 L fermentor tanks of 4 L that cultured secondary seed is inserted liquid amount by 5% inoculum size
33g/L, pH6.0, glycerine are food grade) in, 400 rev/mins of rotating speeds, air flow 2.5vvm.28 ℃ of condition bottom fermentation 48h record that otan content is 122.4 g/L in the fermented liquid.The cultivation of I and II seed is identical with embodiment 1.
Utilize said strain HD 924 ferment glycerins to produce the method for otan, be specially: strain HD 924 is inoculated in the first order seed substratum by 3% inoculum size, and 200 rev/mins, aerobic cultivation is 1 day under 28 ℃ of conditions.Cultured first order seed is inserted in the 250 mL triangular flasks that secondary seed medium is housed (liquid amount is 50 mL) by 5% inoculum size then, by 5% inoculum size cultured secondary seed is inserted in the fermentor tank that liquid amount is 100L behind 8 h and carry out fed-batch fermentation.200 rev/mins of fermentor tank rotating speeds, air flow 1.0 vvm, 28 ℃ of condition bottom fermentation 48h measure otan and residual glycerol content in the fermented liquid.The I and II seed culture medium is formed identical with embodiment 1.
Initial fermentative medium formula: glycerine 50g/L, yeast powder 15g/L, CaCO
31g/L, pH transfers to 6.0.Feed supplement liquid glycerol concentration 100g/L, 124 ℃ of sterilization 40 min are subsequent use.When initial glycerol concentration in the fermented liquid drops to 15g/L when following, begin stream and add and mend feed supplement liquid glycerol concentration in the fermented liquid is controlled at 5～15g/L, used glycerine is food grade.
Measure otan and residual glycerol content in the fermented liquid at fermentation 0h, 2h, 4h, 8h, 16h, 20h, 24h, 28h, 32h, 36h, 40h, 44h, 48h respectively, experimental result is seen table 6.
48 h ferment; Otan content 171.2 g/L in the time of following jar, total is 193.4 g/L after dropping into glycerine (glycerine adds feed supplement glycerine in the initial medium) conversion, residual glycerol 6.4 g/L; (DHA/ drops into glycerine to the otan transformation efficiency, is 88.5% w/w).
Claims (5)
1. a microbial fermentation is produced the strain HD 924 of otan, its minute the generic Acetobacteraceae (
Acetobacteraceae) the gluconobacter suboxydans genus (
Gluconobacter) not the Tuo Shi gluconobacter suboxydans (
Gluconobacter frateurii), this bacterial strain at the preserving number at China Committee for Culture Collection of Microorganisms common micro-organisms center is: CGMCC No. 5397.
2. the method for utilizing said strain HD 924 ferment glycerins of claim 1 to produce otan; It is characterized in that: comprise the steps: strain HD 924 is inserted fermention medium by 1～10% inoculum size, under aerobic condition in 28～30 ℃ of fermentation culture 48～72 h; Said fermention medium consists of: glycerine 50～350g/L, nitrogenous source 5～20g/L, lime carbonate 0.1～15g/L, medium pH 4～7.
3. the method for producing otan according to the said ferment glycerin of claim 2 is characterized in that, strain HD 924 first inoculum sizes by 1～10% is inoculated in the first order seed substratum, in 28～30 ℃ of aerobic cultivation 4～24 h; Then cultured first order seed is inserted in the secondary seed medium by 1～10% inoculum size, behind 8～10 h cultured secondary seed is inserted fermention medium again by 1～10% inoculum size; Wherein, consisting of of said first order seed substratum: glycerine 30～50g/L, yeast powder 5～15g/L, KH
2PO
40.1～0.5g/L, the pH nature; Consisting of of said secondary seed medium: glycerine 30～50g/L, yeast powder 5～15g/L, CaCO
30.1～0.5g/L, pH 4.0～7.0.
4. according to the method for claim 2 or 3 said ferment glycerins production otans, it is characterized in that said glycerine is selected from food grade glycerine, technical grade glycerine or saponification raw glycerine.
5. according to the method for claim 2 or 3 said ferment glycerins production otans, it is characterized in that said nitrogenous source is selected from yeast powder, Carnis Bovis seu Bubali cream, peptone, steeping water, urea or inorganic ammonium salt.
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| CN109576287A (en) * | 2018-11-14 | 2019-04-05 | 河南大学 | CrpMApplication of the gene in the DHA stress tolerance for improving Gluconobacter bacterial strain |
| 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 |
| CN112680386A (en) * | 2021-02-03 | 2021-04-20 | 河南大学 | Application of corn rhizosphere growth-promoting bacteria in promoting plant growth |
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