CN103243031A

CN103243031A - High-permeability-resistant aureobasidium pullulans strain and application thereof

Info

Publication number: CN103243031A
Application number: CN2013101739196A
Authority: CN
Inventors: 陈叶福; 肖冬光; 李军侠; 郭学武; 张翠英; 董健; 杜丽平
Original assignee: Tianjin University of Science and Technology
Current assignee: Tianjin University of Science and Technology
Priority date: 2013-05-10
Filing date: 2013-05-10
Publication date: 2013-08-14
Anticipated expiration: 2033-05-10
Also published as: CN103243031B

Abstract

The invention discloses a high-permeability-resistant aureobasidium pullulans strain. The aureobasidium pullulans strain (Aureobasidium pullulans) ER35 is collected in the China General Microbiological Culture Collection Center (CGMCC) on 18, Jan in 2013, and the collection number is CGMCC No.7152. The strain can take xylose as a carbon source for performing fermentation production on erythritol, fewer by-products are produced, and a small amount of xylitol and maltitol is produced only. The erythritol is subjected to fermentation production by utilizing the strain, the erythritol is high in yield, the utilization rate of the xylose is improved, the renewable resource hemicellulose (xylose) is fully utilized, novel raw materials are provided for production and development of erythritol production, and the raw material utilization rate of the lignocellulose biotransformation process can be effectively improved. A novel way is developed for reasonable and effective utilization of xylose.

Description

The anti-height of one strain oozes Aureobasidium pullulans and application thereof

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 erythritol thereof.

Background technology

Industrial erythritol mainly is to utilize the starch material fermentative production with osmophilic yeast and other microorganisms that produces erythritol.The bacterial classification that can produce erythritol has candida magnoliae (Candida magnoliae), torulopsis (Torulopsis sp.), short stalk mould Pseudomonas (Aureobasidium sp.), Ye Luoweiya yeast (Yarrowia lipolytica), and torulopsis (Torulopsis), trichosporon (Trichosporum), Trigonopsis (Trigonopsis), Pichia (Pichia) etc. also can be produced erythritol in addition.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 erythritol, and domestic and in the world the bacterial classification that adopts of suitability for industrialized production mainly also be the yeast that anti-height oozes.

Chinese patent application 201210187067.1 discloses a strain and has produced the bacterial strain of erythritol and produce the method for erythritol with it, invention provides a strain to produce the bacterial strain of erythritol, its classification called after candiyeast (Candida sp.) SK25.001, be preserved in Chinese typical culture collection center, deposit number is CCTCC No:M2011477.Comprise with its method of producing erythritol: (a) in containing the fermention medium of glucose, utilize this bacterial classification to ferment; (b) separation and purification erythritol from the gained fermented liquid.With candiyeast provided by the invention (Candida sp.) CCTCC No:M2011477 bacterial strain, in the fermention medium that with glucose is carbon source through the fermentation of 4d, residual sugar can reach below 10%, erythritol concentration in the fermented liquid reaches 10%-13%, for the preparation of industrialization of erythritol provides new method.

Chinese patent 200510102929.6 discloses a kind of Candida lipolytica bacterium and has produced the method for erythritol with it, this patent relates to the microbial fermentation field, be specifically related to the Candida lipolytica (Candida lipolytica) of high yield erythritol and utilize this yeast strain to produce the method for erythritol, this method comprises: (A) utilize the Candida lipolytica of high yield erythritol to ferment in containing the fermention medium of glucose, to obtain containing the fermented liquid of erythritol; (B) separation and purification erythritol from above-mentioned fermented liquid.

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, it is the monose that occurring in nature content is only second to glucose, therefore seek the natural microbial of wood-sugar fermentation and seed selection efficiently the wood-sugar fermentation bacterial strain become the research focus.Find more than 100 kind of microorganism energy metabolism wood sugar up to now, comprised bacterium, fungi and yeast, be mainly used to produce alcohol fuel.

Summary of the invention

The invention provides the anti-height of a strain and ooze Aureobasidium pullulans, but this bacterial strain xylose-fermenting is effectively produced erythritol.

Technical scheme of the present invention:

The anti-height of one strain oozes Aureobasidium pullulans ER35, described Aureobasidium pullulans (Aureobasidium pullulans) ER35 is preserved in Chinese microorganism strain management committee common micro-organisms center (CGMCC) on January 18th, 2013, preserving number is CGMCC No.7152, preservation address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode 100101.

It is the fermention medium fermentative production erythritol of carbon source with the wood sugar that anti-height oozes out the short mould ER35 of bud, and anti-height oozes out the short just mould ER35 fermentation of bud erythritol output can reach 26g/L-34g/L, and transformation efficiency reaches 25%-42%;

Described fermention medium is main carbon source with wood sugar, adds other nitrogenous sources and trace element composition;

Preferred fermention medium consists of:

Wood sugar 80-160g/L, yeast soak powder 15-30g/L, KH ₂PO ₄0.2-1g/L, ZnSO ₄7H ₂O0.5-1.5g/L, L-tyrosine 2-10g/L, citric acid 2-10g/L.

Wood sugar can replace with xylose mother liquid in the described fermention medium, and composition (mass percent) is as follows: wood sugar 35-40%, and 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.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 the characteristic screening by ultraviolet mutagenesis in conjunction with anti-height and is obtained the Aureobasidium pullulans mutant strain ER35 that anti-height oozes, can improve erythritol output, and by fermention medium and fermentation condition optimization, effectively improved the erythritol output when this mutant strain is carbon source with the wood sugar.Using bacterial classification of the present invention and wood sugar effectively can be converted into erythritol, is that carbon source through fermentation erythritol output can reach 31.42g/L and 26.35g/L with wood sugar and xylose mother liquid respectively, and transformation efficiency is respectively 38.59% and 25.36%; 5L fermentor tank wood sugar and xylose mother liquid fermentation erythritol output can reach 33.78g/L and 28.46g/L respectively, transformation efficiency is respectively 41.75% and 28.43% the present invention and has opened up new way for effective utilization of wood sugar, also for erythritol production exploitation provides new raw material, can effectively improve the raw material availability of lignocellulose biotransformation.

Embodiment

Embodiment 1: anti-height oozes, can utilize the mutagenesis screening of the Aureobasidium pullulans mutant strain ER35 of xylose production erythritol

Be starting strain with Aureobasidium pullulans (Aureobasidium pullulans) CGMCC3.00837 that can utilize wood sugar, 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 big behind 30 ℃ of lucifuges cultivation 2-3d is connected on the TTC solid plate, obtain the deep mutant strain of 120 strain colors at last, called after ER1-ER120.Mutant strain is carried out the erythritol fermenting experiment, measure fermented liquid mesoerythrit content behind the fermentation 144h, do contrast with the strain of setting out, obtain the mutant strain ER35 that a strain erythritol output obviously improves, erythritol output improves 50.92% than starting strain; Transformation efficiency improves 20.71%, the results are shown in Table 1.Starting strain and mutant strain ER35 erythritol tolerance situation see Table 2.

Table 1: the contrast of erythritol high productive mutant and starting strain

Table 2 starting strain and mutant strain ER35 erythritol 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

Being experimental strain with mutant strain ER35, is carbon source with the wood sugar, and by substratum and fermentation condition optimization, the wood-sugar fermentation substratum 1 that is optimized is: wood sugar 120g/L, yeast soak powder 20g/L, KH ₂PO ₄0.5g/L, ZnSO ₄7H ₂O1g/L, L-tyrosinase 15 g/L, citric acid 5g/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.Also can select wood-sugar fermentation substratum 2, the preparation method is as follows: with about 4 times of xylose mother liquid dilutions, making its xylose concentration is 121g/l, contain glucose 44g/L, pectinose 46g/L, semi-lactosi 18g/L simultaneously, 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 ER35 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.Primary seed solution with 10% inoculum size, is inserted in the secondary seed medium, and 30 ℃, 180r/min shaking table are cultivated 14h, get secondary 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 inserts in above-mentioned wood-sugar fermentation substratum 1 or 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 ER35 shake flask fermentation experimental result

As shown in Table 3, mutant strain ER35 can effectively utilize the xylose production erythritol, though also have a small amount of wood sugar not to be utilized, the utilization ratio of wood sugar is still than higher than the bacterial strain of other production erythritol.

Embodiment 3:5L ferment tank

On the basis of shake flask fermentation, carry out the experiment of ER35 xylose fermentation for producing erythritol by the 5L fermentor tank, investigated the fermentor cultivation mesoerythrit and produced and thalline biomass situation.Seed culture medium and seed culture method, wood-sugar fermentation substratum 1 and 2 form with embodiment 2 in identical.

5L ferment tank condition is specially: the fermentor tank liquid amount is 60% of jar appearance, and inoculum size 10%, initial pH value are 6.5, and leavening temperature is 30 ℃, and dissolved oxygen is 60%, fermentation 120h.Erythritol output, residual sugar amount and biomass are measured in certain hour sampling at interval, the results are shown in Table 3.

Table 3. mutant strain ER355L jar fermenting experiment result

On the jar of 5L, wood sugar substratum 1 and 2 fermentation erythritol output and transformation efficiency all increase with respect to shaking bottle, and the product formation time shortens, and thalli growth is very fast, for follow-up study is laid a good foundation.

Claims

1. the anti-height of a strain oozes Aureobasidium pullulans ER35, it is characterized in that, described Aureobasidium pullulans (Aureobasidium pullulans) ER35, preserving number is CGMCC No.7152, it is the fermention medium fermentative production erythritol of carbon source that described Aureobasidium pullulans ER35 utilizes with the wood sugar.

2. ooze Aureobasidium pullulans ER35 according to the described anti-height of claim 1, it is characterized in that, fermentative production erythritol output reached 26g/L-34g/L when described anti-height oozed Aureobasidium pullulans ER35 and is carbon source with the wood sugar, and transformation efficiency reaches 25%-42%.

3. ooze Aureobasidium pullulans ER35 according to the described anti-height of claim 1, it is characterized in that: described fermention medium consists of: wood sugar 80-160g/L, yeast soak powder 15-30g/L, KH ₂PO ₄0.2-1g/L, ZnSO ₄7H ₂O0.5-1.5g/L, L-tyrosine 2-10g/L, citric acid 2-10g/L.

4. ooze Aureobasidium pullulans ER35 according to the anti-height of the described strain of the arbitrary claim of claim 1-3, it is characterized in that, described fermention medium is selected 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. the described anti-height of the arbitrary claim of claim 1-3 oozes the application of Aureobasidium pullulans ER35 in the fermentative production erythritol.