CN103305427A - Aureobasidium pullulans mutant strain of high-yield xylitol and application thereof - Google Patents
Aureobasidium pullulans mutant strain of high-yield xylitol and application thereof Download PDFInfo
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- CN103305427A CN103305427A CN2013101732125A CN201310173212A CN103305427A CN 103305427 A CN103305427 A CN 103305427A CN 2013101732125 A CN2013101732125 A CN 2013101732125A CN 201310173212 A CN201310173212 A CN 201310173212A CN 103305427 A CN103305427 A CN 103305427A
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Abstract
The invention discloses a hyperosmotic-resistant aureobasidium pullulans strain and an application thereof in producing xylitol. The Aureobasidium pullulans EK13 is preserved in China General Microbiological Culture Collection Center (CGMCC) on January 18th, 2013, and the preservation Number is CGMCCNo.7153. The strain can be used for producing xylitol through fermenting by taking xylose or xylose mother liquor as a carbon source, and no by-product is produced. Xylitol is produced by using the strain through fermentation, so that the full utilization of a renewable resource hemicellulose (xylose) is realized, the utilization rate of xylose is improved, and a new way is created for the efficient utilization of xylose.
Description
Technical field
The invention belongs to the microbial fermentation technology field, be specifically related to the anti-height of a strain and ooze Aureobasidium pullulans and the application in producing Xylitol thereof.
Background technology
Xylitol (Xylitol) claims again pentitol, is a kind of five-carbon sugar alcohol, and its molecular formula is C
5H
12O
5, molecular weight 152.15.The physico-chemical property of Xylitol is similar to sucrose, sugariness between 0.65～1.05,1.0 suitable with sucrose; It is the sweetest a kind of in the sugar alcohol, and no impurity taste behind the entrance is refrigerant like peppermint; Water-soluble, calorific value is 16.79J/g, is lower than sucrose, is a kind of low-yield sweeting agent, Heat stability is good; Extensively be present in the various fruits and vegetables, such as grass poison, Ge Ba, green plum, spinach and mushroom etc., but content only is 0.014-0.09mg/100g; Birch, to be rich in the pentosan plant material by tree, corn cob, bagasse, rice husk etc. be its good source.These characteristics that Xylitol has make it be widely used in the various fields such as food, medicine, leather, chemical industry, and development prospect is very wide, enjoys attention both domestic and external.
Find according to existing research, yeast is than being easier to wood sugar to be transformed the bacterial strain that generates Xylitol.The more superior bacterial strain of common product Xylitol mainly concentrates on Candida and belongs to (mycocandida), such as C.guilliermondii, and C.tropicalis, C.monii, C.parasilosis; Part belongs to Debaryomyces and belongs to (Debaryomyces), such as D.hansenii and Pachysolen(pipe capsule yeast belong), such as P.tannophilus.These yeast mainly are under the effect of Xylose reductase in its body, take NADPH or NADH as coenzyme, the D-wood sugar are reduced to Xylitol.Because yeast has the good characteristic that anti-height oozes environment, be suitable under the condition of high concentration sugar, growing, so osmophilic yeast is the desirable bacterial classification of producing Xylitol, the research discovery is arranged along with the increase of xylose concentration, the generation speed of Xylitol also increases thereupon.
Chinese patent application 200910092915.9 discloses a kind of preparation method of Xylitol, invention relates to a kind of preparation method of Xylitol, comprise the steps: to be inoculated into through the Xylitol barms of cultivating in the fermention medium in the fermentor tank and cultivate, carry out aerobic, two sections combined fermentations of little oxygen, sectional temperature-controlled in the fermenting process, take the means of segmentation regulation and control in the dissolved oxygen demand, produce Xylitol so that Xylitol is produced bacterial classification metabolism under little good oxygen condition.Xylitol preparation method of the present invention adopts sectionalized ventilation and thermophilic fermentation, has saved sterile air and cooling water amount, has reduced cost; Production process is simple, safe, energy-conservation; And avoided the use of the nickel catalyzator in the traditional method, protected environment.
Chinese patent application: 200910087819.5, name is called a kind of method of utilizing xylose mother liquid to produce Xylitol and L-arabinose.Invention relates to a kind of method of utilizing xylose mother liquid to produce Xylitol and L-arabinose, it utilizes Xylitol to produce the strain fermentation xylose mother liquid, then remove condensing crystal behind thalline, pigment and the impurity in the fermented liquid, obtain xylitol crystal and Xylitol mother liquor, wherein Xylitol mother liquor obtains the L-arabinose phase through the chromatographic system separation, and L-arabinose phase condensing crystal can access the L-arabinose crystal.It is that raw material is produced Xylitol and L-arabinose simultaneously that the inventive method takes full advantage of the trade waste xylose mother liquid, at first utilize the synthetic Xylitol of xylose and glucose fermentation in the xylose mother liquid, not only increase the output of producing Xylitol, also can reduce the difficulty of therefrom separating L-arabinose.The inventive method starting material wide material sources and cheap, therefore economize on resources and production cost low, can reduce the environmental pollution that trade waste causes simultaneously.
Lignocellulose raw material is renewable resources abundant, the most cheap on the earth, and the trans-utilization of wood fibre resource has become inexorable trend.Hemicellulose accounts for the 20%-35% of lignocellulose raw material, main monose in its hydrolyzed solution is wood sugar, therefore it is the monose that occurring in nature content is only second to glucose, seeks the natural microbial of wood-sugar fermentation and the wood-sugar fermentation bacterial strain of breeding high-efficiency becomes study hotspot.Find up to now 100 multiple-microorganism energy metabolism wood sugars, comprised bacterium, fungi and yeasts, be mainly used to produce alcohol fuel.
The present invention is can also producing the Aureobasidium pullulans of Xylitol as starting strain by fermenting xylose, having obtained a strain through mutagenesis screening can utilize wood sugar effectively to produce the mutant strain of Xylitol, realized taking full advantage of of renewable origin resource hemicellulose (wood sugar), for new way has been opened up in effective utilization of wood sugar.
Summary of the invention
The anti-height that the invention provides plant height product Xylitol oozes Aureobasidium pullulans and application thereof, but this bacterial strain xylose-fermenting is effectively produced Xylitol.
Technical scheme of the present invention:
One strain Aureobasidium pullulans EK13, described Aureobasidium pullulans (Aureobasidium pullulans) but the EK13 xylose-fermenting is produced Xylitol, this bacterial strain is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC) on January 18th, 2013, preserving number is CGMCC No.7153, the preservation address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode 100101.
Anti-height oozes out the short just mould EK13 xylose-fermenting alcohol of bud output and reaches 12g/L-63g/L, and transformation efficiency is 18-73%.
A kind of method of using the above-mentioned short just mould EK13 production Xylitol that sprouts, step is:
(1) seed culture: slant strains access seed culture medium is cultivated the acquisition seed liquor step by step;
(2) fermentation culture: the inoculum size with 5～15%, the fermentation of access wood-sugar fermentation substratum, the wood-sugar fermentation substratum is take wood sugar as main carbon source.
The shaker fermentation condition is: liquid amount 10-30%, shaking speed 140～180r/min, 26～32 ℃ of temperature, fermentation time are 96～144h.
The ferment tank condition is: liquid amount 20-60%, dissolved oxygen are 55%～75%, cultivate 24～96h under the condition of 26～32 ℃ of temperature, pH value 5～8.
Consisting of of described seed culture medium: wood sugar 20g/L, peptone 20g/L, yeast soak powder 10g/L.
Consisting of of described wood-sugar fermentation substratum: wood sugar (80～160) g/L, yeast soak powder (2～8) g/L, urea element (0.5～1.5) g/L, (NH
4) 2SO4(1～3) g/L.
Described fermention medium preferably uses the xylose mother liquid substratum, consists of: with the wood sugar in the xylose mother liquid replacement fermention medium, described xylose mother liquid composition is as follows: wood sugar 35-40%, pectinose 8-16%, semi-lactosi 9-15%, glucose 8-11%, all the other are water; The total sugar content that is diluted in the xylose mother liquid substratum is 50g/L.
Described substratum adds yeast by xylose mother liquid by dilution and soaks powder, urea and ammonium sulfate gained.
Xylose mother liquid is the tankage of xylose production, and xylose mother liquid is the waste liquid that stays behind the xylose crystalline in the xylose production process.
Beneficial effect:
The present invention is oozed characteristic screening by ultraviolet mutagenesis in conjunction with anti-height and is obtained that anti-height oozes, the Aureobasidium pullulans mutant strain EK13 of Xylitol output increased, and by optimization of fermentation conditions, Effective Raise the Xylitol output of this mutant strain during take wood sugar as carbon source.Use bacterial classification of the present invention and wood sugar effectively can be converted into Xylitol, wood sugar and xylose mother liquid shake flask fermentation Xylitol output can reach 63.42g/L and 14.31g/L respectively, and transformation efficiency is respectively 73.26% and 21.45%; 5L fermentor tank wood sugar and xylose mother liquid xylose-fermenting alcohol output can reach 49.34g/L and 12.46g/L respectively, and transformation efficiency is respectively 61.75% and 18.43%.The present invention is that new way has been opened up in effective utilization of wood sugar, also for the Xylitol production development provides new raw material, but the raw material availability of Effective Raise lignocellulose biotransformation.
Embodiment
Embodiment 1: anti-height oozes, can utilize the mutagenesis screening of the Aureobasidium pullulans mutant strain EK13 of xylose production erythritol
Take Aureobasidium pullulans (Aureobasidium pullulans) CGMCC3.00837 that can utilize wood sugar as starting strain, selecting lethality rate is that 85% ultraviolet mutagenesis dosage (irradiation 240s) carries out mutagenesis to Aureobasidium pullulans, bacterium liquid after the mutagenesis is evenly coated the height that contains the 30g/L wood sugar to be oozed on the flat board, selecting single bacterium colony point that growth is fast, bacterium colony is large behind 30 ℃ of lucifuges cultivation 2～3d is connected on the TTC solid plate, obtain at last the deep mutant strain of 120 strain colors, called after EK1～EK120.Mutant strain is carried out the xylitol fermentation experiment, measure Determination of Xylitol in the fermented liquid behind the fermentation 144h, do contrast with the strain of setting out, obtain the mutant strain EK13 that a strain Xylitol output obviously improves, Xylitol output has improved 1 times than starting strain; Transformation efficiency improves 37.71%, the results are shown in Table 1.
Table 1: the contrast of Xylitol high productive mutant and starting strain
Table 2: starting strain and mutant strain EK13 wood sugar tolerance situation
" +++" the expression well-grown; " ++ " expression growth is general; A little less than "+" expression growth; "-" expression is not grown
Embodiment 2: shake flask fermentation
Take mutant strain EK13 as experimental strain, take wood sugar as carbon source, by substratum and fermentation condition optimization, the wood-sugar fermentation substratum 1 that is optimized is: wood sugar 120g/L, yeast soak powder 5g/L, urea element 1g/L, (NH
4)
2SO
42g/L, the 0.1MPa 15min that sterilizes; Optimization of fermentation conditions is: inoculum size 10%, and liquid amount 50mL/250mL, shaking speed 180r/min, 30 ℃ of temperature, pH value 6.5, incubation time is 144h.The preparation method of wood-sugar fermentation substratum 2 is as follows: with about 4 times of xylose mother liquid dilutions, making its xylose concentration is 121g/L, contain simultaneously glucose 44g/L, pectinose 46g/L, semi-lactosi 18g/L, according to the composition of described wood-sugar fermentation substratum 1, add other medium components except wood sugar, glucose, pectinose and semi-lactosi.
Picking one ring EK13 thalline is seeded to the 250mL triangular flask that 50mL first order seed substratum is housed and cultivates from activated inclined plane, and 30 ℃ of 180r/min shaking tables are cultivated 12h, get primary seed solution.With 10% inoculum size, in the access secondary seed medium, 30 ℃, 180r/min shaking table are cultivated 10h, get secondary seed solution with primary seed solution; The I and II seed culture medium consists of: wood sugar 20g/L, peptone 20g/L, yeast soak powder 10g/L.Seed liquor accesses in above-mentioned wood-sugar fermentation substratum 1 and 2 with 10% inoculum size, and 30 ℃ of 180r/min shake flask fermentations are cultivated 144h, the results are shown in Table 3.
Table 3. mutant strain EK13 shake flask fermentation experimental result
As shown in Table 3, mutant strain EK13 can efficiently utilize the xylose production Xylitol, although also have a small amount of wood sugar not to be utilized, but the utilization ratio of wood sugar is still higher than the bacterial strain of other production Xylitol, next step can further be studied this bacterial strain, improve its xylose utilization rate, make more wood sugar can be converted into Xylitol.
Embodiment 3:5L ferment tank
On the basis of shake flask fermentation, carry out the experiment of EK13 xylose fermentation for producing Xylitol by the 5L fermentor tank, investigated Xylitol production and thalline biomass situation in the fermentor cultivation.Seed culture medium and seed culture method, Xylose fermention medium form with embodiment 2 in identical.
5L ferment tank condition is specially: the fermentor tank liquid amount is 60% of tank appearance, and inoculum size 10%, initial pH value are 6.5, and leavening temperature is 30 ℃, and dissolved oxygen is 60%, fermentation 96h.Xylitol output, residual sugar amount and biomass are measured in interval certain hour sampling, the results are shown in Table 4.
Table 4 mutant strain EK13 5L tank fermenting experiment result
On the tank of 5L, wood sugar and xylose mother liquid xylose-fermenting alcohol output and transformation efficiency are all more stable with respect to shaking flask, and the product formation time shortens, and thalli growth is very fast, for follow-up study is laid a good foundation.
Claims (5)
1. a plant height produces Xylitol Aureobasidium pullulans EK13, it is characterized in that: described Aureobasidium pullulans (Aureobasidium pullulans) EK13, deposit number is CGMCC No.7153, and the fermention medium of this bacterial strain take wood sugar as main carbon source produced Xylitol.
2. a described plant height produces Xylitol Aureobasidium pullulans EK13 according to claim 1, it is characterized in that: described Aureobasidium pullulans EK13 xylose-fermenting produces Xylitol output and reaches 12g/L-63g/L, and transformation efficiency is 18-73%.
3. a described plant height produces Xylitol Aureobasidium pullulans EK13 according to claim 1, and it is characterized in that: described fermention medium consists of: wood sugar 80～160g/L, yeast soak powder 2～8g/L, urea element 0.5～1.5g/L, (NH
4)
2SO
41～3g/L.
4. a described plant height produces Xylitol Aureobasidium pullulans EK13 according to claim 1, it is characterized in that: described fermention medium uses the xylose mother liquid substratum, consist of: with the wood sugar in the xylose mother liquid replacement fermention medium, described xylose mother liquid composition is as follows: wood sugar 35-40%, pectinose 8-16%, semi-lactosi 9-15%, glucose 8-11%, all the other are water; The total sugar content that is diluted in the xylose mother liquid substratum is 50g/L.
5. a described plant height produces the application of Xylitol Aureobasidium pullulans EK13 in xylitol zymolysis production according to claim 1.
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| CN101988036A (en) * | 2010-11-01 | 2011-03-23 | 吉林农业大学 | Aureobasidium pullulans strain as well as preparation method and application thereof in producing pigment-free pullulan |
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| CN101988036A (en) * | 2010-11-01 | 2011-03-23 | 吉林农业大学 | Aureobasidium pullulans strain as well as preparation method and application thereof in producing pigment-free pullulan |
Non-Patent Citations (2)
| Title |
|---|
| 崔堂兵等: "出芽短梗霉的研究进展", 《工业微生物》, vol. 32, no. 2, 30 June 2002 (2002-06-30) * |
| 段效辉等: "普鲁兰多糖高产菌株Y68葡萄糖基转移酶的纯化及特性研究", 《微生物学杂志》, vol. 30, no. 6, 30 November 2010 (2010-11-30) * |
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