CN103290068A - Method for producing erythritol by utilizing hypertonicity-resistant Aureobasidium pullulans - Google Patents
Method for producing erythritol by utilizing hypertonicity-resistant Aureobasidium pullulans Download PDFInfo
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Abstract
The invention discloses a method for producing erythritol by utilizing a strain of hypertonicity-resistant Aureobasidium pullulans. The Aureobasidium pullulans ER35 is preserved in China General Microbiological Culture Collection Center (CGMCC) in Jan 1st, 2013, and the preservation number of the Aureobasidium pullulans ER35 is CGMCC No.7152. The strain can be applied to fermentation production of the erythritol by taking xylose or xylose mother solution as a carbon source, and by-products are less and only include small amounts of xylitol and maltitol. By fermenting and producing the erythritol by utilizing the strain, regenerative resource hemicelluloses (xylose) is adequately utilized, the utilization capacity of the xylose is improved, and a new way for reasonably and effectively utilizing the xylose is opened up.
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 production erythritol.
Background technology
Erythritol (Erythritol), its chemistry 1 by name, molecular formula is C
4H
10O
4, molecular weight 122.12,126 ℃ of fusing points, 329 ℃～331 ℃ of boiling points, outward appearance is white crystalline powder, and is comminuted good, and water-soluble meeting absorbs more energy, and solution heat is-97.4J/g.It is present popular a kind of filled-type polyalcohols sweeting agent in the world, have the taste that exactly likes sucrose and thermal value close to zero, sugariness is the 60%-70% of sucrose.Place of sucrose is made low-calorie healthcare products, and it is edible to be suitable for obesity, hypertension and cardiovascular patient.It also has characteristics such as energy is low, physiological tolerance is high, freezing point is low, heat-resisting acid resistance strong, prevention of dental caries in addition, and in addition because erythritol is not degraded by enzyme, therefore involved in sugar metabolism and change of blood sugar are not suitable for patients with diabetes mellitus.These characteristics that erythritol has make it be widely used in food, medicine and fields such as healthcare products production and chemical industry, and development prospect is very wide, enjoys attention both domestic and external.
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.
The present invention is starting strain with the Aureobasidium pullulans of can the metabolism wood sugar and can produce erythritol, having obtained a strain through mutagenesis screening can utilize wood sugar effectively to produce the mutant strain of erythritol, 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 invention provides anti-height and ooze the method that the Aureobasidium pullulans xylose-fermenting is produced erythritol.
Technical scheme of the present invention:
A kind of method of utilizing Aureobasidium pullulans ER35 to produce erythritol, adopting with wood sugar is the fermention medium fermentative production of main carbon source, step is:
(1) seed culture: slant strains switching seed culture medium is cultivated the acquisition seed liquor step by step;
(2) fermentation culture: seed liquor is inserted fermentation culture in the fermention medium; Fermentation culture is carried out in shaking table or fermentor tank; The shaker fermentation condition is: fermention medium loading amount 10-30%, and shaking speed 140～180r/min, 26～32 ℃ of temperature, fermentation time are 96～144h; The fermentor cultivation condition is: inoculum size 5～15%, fermention medium loading amount 30-70%: be 55%～75% at dissolved oxygen, cultivate 96～120h under the condition of 26～32 ℃ of temperature, pH value 5～8.
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.
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's postcode 100101.
It can wood sugar be that carbon source through fermentation is produced erythritol that anti-height oozes Aureobasidium pullulans ER35, 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%;
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, erythritol output improves, 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 respectively 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.
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; "-" represents not grow 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.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 glucose 44g/L, pectinose 46g/L, semi-lactosi 18g/L simultaneously, according to the composition of described wood-sugar fermentation substratum, add other medium components except wood sugar, glucose, pectinose and semi-lactosi.Xylose mother liquid provides for Long Li company of xylose production factory or according to the prior art for preparing xylose mother liquid.
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 2, 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 4.
Table 4. 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 (5)
1. one kind is utilized anti-height to ooze out the method that the short just mould ER35 of bud produces erythritol, comprises the steps:
(1) seed culture: cultivate step by step behind the slant strains switching seed culture medium and obtain seed liquor;
(2) fermentation culture: seed liquor is inserted fermentation culture in the fermention medium; Fermentation culture is carried out in shaking table or fermentor tank; Described fermention medium is main carbon source with wood sugar; Described anti-height oozes Aureobasidium pullulans (Aureobasidium pullulans) ER35, and preserving number is CGMCC No.7152.
2. according to the described a kind of method of utilizing anti-height to ooze Aureobasidium pullulans ER35 production erythritol of claim 1, it is characterized in that, described shaker fermentation condition is: fermention medium loading amount 10-30%, shaking speed 140～180r/min, 26～32 ℃ of temperature, fermentation time are 96～144h.
3. according to the described a kind of method of utilizing anti-height to ooze Aureobasidium pullulans ER35 production erythritol of claim 1, it is characterized in that, described fermentor cultivation condition is: inoculum size 5～15%, fermention medium loading amount 30-70% is 55%～75% at dissolved oxygen, cultivates 96～120h under the condition of 26～32 ℃ of temperature, pH value 5～8.
4. according to the described a kind of method of utilizing anti-height to ooze Aureobasidium pullulans ER35 production erythritol of the arbitrary claim of claim 1-3, 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.
5. according to the described a kind of method of utilizing anti-height to ooze Aureobasidium pullulans ER35 production erythritol of claim 4, it is characterized in that, described fermention medium is selected following composition: 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.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109504733A (en) * | 2018-12-18 | 2019-03-22 | 浙江华康药业股份有限公司 | A kind of preparation method of antierythrite |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4939091A (en) * | 1986-09-09 | 1990-07-03 | Director Of National Food Research Institute, Ministry Of Agriculture, Forestry And Fisheries | Novel auerobasidium sp. microorganisms, method for obtaining the same and method for preparing erythritol with the same |
| CN102876732A (en) * | 2012-09-19 | 2013-01-16 | 北京化工大学 | Method for preparing high-added-value sugar alcohols by efficiently using wood fiber raw materials |
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2013
- 2013-05-10 CN CN201310172815.3A patent/CN103290068B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4939091A (en) * | 1986-09-09 | 1990-07-03 | Director Of National Food Research Institute, Ministry Of Agriculture, Forestry And Fisheries | Novel auerobasidium sp. microorganisms, method for obtaining the same and method for preparing erythritol with the same |
| CN102876732A (en) * | 2012-09-19 | 2013-01-16 | 北京化工大学 | Method for preparing high-added-value sugar alcohols by efficiently using wood fiber raw materials |
Non-Patent Citations (2)
| Title |
|---|
| HIROAKI ISHIZUKA，ET AL.: "Breeding of a Mutant of Aureobasidium sp. with High Erythritol Production", 《JOURNAL OF FERMENTATION AND BIOENGINEERING》, vol. 68, no. 5, 31 December 1989 (1989-12-31), pages 310 - 314 * |
| 叶娴等: "赤藓糖醇高产菌株的筛选、鉴定及发酵特性的初步研究", 《生物技术》, vol. 17, no. 5, 31 October 2007 (2007-10-31), pages 54 - 57 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109504733A (en) * | 2018-12-18 | 2019-03-22 | 浙江华康药业股份有限公司 | A kind of preparation method of antierythrite |
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