CN103981224B - D-R alcohol production method based on multiple-shaped nuohan inferior yeast - Google Patents
D-R alcohol production method based on multiple-shaped nuohan inferior yeast Download PDFInfo
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- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 6
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- 229920001817 Agar Polymers 0.000 claims description 4
- 239000008272 agar Substances 0.000 claims description 4
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- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 3
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- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 3
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 3
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 3
- HEBKCHPVOIAQTA-NGQZWQHPSA-N d-xylitol Chemical compound OC[C@H](O)C(O)[C@H](O)CO HEBKCHPVOIAQTA-NGQZWQHPSA-N 0.000 abstract description 22
- HEBKCHPVOIAQTA-QWWZWVQMSA-N D-arabinitol Chemical compound OC[C@@H](O)C(O)[C@H](O)CO HEBKCHPVOIAQTA-QWWZWVQMSA-N 0.000 abstract description 19
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Abstract
The present invention provides a kind of D 1,2,3,4,5-pentanepentol production method based on multiple-shaped nuohan inferior yeast: carry out fermentation seed cultivation after being tamed under the conditions of height oozes by multiple-shaped nuohan inferior yeast, then fermentation medium it is seeded to, first 18～24h are cultivated at 30～38 DEG C, then 40～49 DEG C are continued to cultivate 18～24h, last 28～38 DEG C are continued to cultivate 74～96h, the present invention is by comprehensive strain domestication and the technique of regulation and control fermentation temperature, improve yeast production D arabitol ability, dry cell weight and D 1,2,3,4,5-pentanepentol yield are respectively up to 5.26g/L and 114.92g/L, show that the present invention can be used for improving the yield of multiple-shaped nuohan inferior yeast fermenting and producing D 1,2,3,4,5-pentanepentol；And the method for the invention is easily operated, cost is relatively low, be readily applied to produce D 1,2,3,4,5-pentanepentol fermentation process in.
Description
Technical field
The present invention relates to technical field of biological fermentation, particularly to a kind of multiple-shaped nuohan inferior yeast (Hansenula
Polymorpha, H.polymorpha) method of DL-1 fermenting and producing D-R alcohol.
Background technology
D-R alcohol (D-arabitol) is a kind of five carbon multi-sugar alcohols.Its molecular formula is C5H12O5,
Relative molecular weight is 152.12, and fusing point is 103 DEG C, and optical rotation is 130 DEG C, is white column knot under room temperature
Crystalline substance, soluble in water, insoluble in ether.Multi-sugar alcohol refers to that the carbonyl of sugar is many through the class being hydrogenated to
Unit's alcohol.D-R alcohol has in fields such as medicine, food, chemical industry and is increasingly widely applied.
D-arabitol is primarily present in mushroom class and lichens biology, but content is the lowest, it is impossible to meet day by day
The market demand increased.
At present, the production method of D-arabitol is mainly produced by chemical synthesis, will arabinose
Catalytic hydrogenation obtains D-R alcohol at high temperature under high pressure.This method needs to enter at high temperature under high pressure
Row reaction, requires higher to appointed condition, and is hydrogenated with and does not has selectivity, and the impurity of generation is more, leads
Cause subsequent extracted separating technology loaded down with trivial details.Additionally, the method production cost is high, seriously polluted.
The shortcoming existed in view of chemical synthesis process, sight is gradually transferred to biological fermentation process by people.Raw
The advantages such as it is relatively low that thing fermentation method has cost, it is easy to high density fermentation, energy-conserving and environment-protective.Biological fermentation process is raw
The bacterial strain producing D-R alcohol conventional is typically all osmophilic yeast.This exists mainly due to osmophilic yeast
Height oozes in environment metabolism can produce multi-sugar alcohol, it is achieved protection is in the cell of growth under the conditions of height oozes
Purpose.Be currently used for produce D-R alcohol bacterial strain be mainly derived from Hansenula (Hansenula),
Pichia (Pichia), mycocandida (candida), zygosaccharomyces belong to (Zygosaccharomyces)
With Debaryomyces (Debaryomyces).
Multiple-shaped nuohan inferior yeast (Hansenula polymorpha) in Hansenula belongs to heat-resistant yeast,
The highest can grow under the conditions of 49 DEG C, it is easy to high density fermentation.It should be noted that of a relatively high training
Foster temperature advantageously reduces the probability of pollution microbes in large scale fermentation incubation.Additionally, the multiform Chinese
Inferior yeast is a kind of facultative aerobic yeast, and the suitableeest scope of pH is 2.5～8.0, can be cheap non-selective
Growing in culture medium, production cost is relatively low.Therefore, multiple-shaped nuohan inferior yeast is current internationally recognized ideal
One of cell factory, can be widely used for producing the food and medicine product of high added value.
Except indivedual researcheres utilize yeast to carry out glucose converting production D-R alcohol, convert effect
Rate can reach 50～60% (Jiangning etc., patent of invention, CN1286306A) beyond, current existing utilization
It is 30～40% that biological fermentation process produces the inversion rate of glucose of D-R alcohol, and this is to biological sugar alcohol industry
Great significance.But still suffering from the shortcomings such as sterility requirements is high, later stage separation requirement is high, this also limits
The development of biological sugar alcohol.Change ferment additionally, current biotransformation method underuses fermentation condition
The impact of blast cell metabolic mechanism is to reach to improve the purpose of D-R alcohol yield.
Summary of the invention
It is an object of the invention to provide a kind of D-R alcohol producer based on multiple-shaped nuohan inferior yeast
Method.
For reaching above-mentioned purpose, present invention employs techniques below scheme.
Carry out fermentation seed after being tamed under the conditions of height oozes by multiple-shaped nuohan inferior yeast and cultivate to obtain fermentation seed cultivation
Liquid, carries out fermentation culture after fermentation seed culture fluid is seeded to fermentation medium, the process of fermentation culture
For: first in 30～38 DEG C of shaken cultivation 18～24h, then cultivation temperature is risen a height of 40～49 DEG C of continuation
Shaken cultivation 18～24h, is then down to cultivation temperature 28～38 DEG C and continues shaken cultivation 74～96h, fermentation
In incubation, shaking speed remains 160～200r/min.
The source of described multiple-shaped nuohan inferior yeast is ATCC No.26012.
The acclimation method of described multiple-shaped nuohan inferior yeast is: by the multiple-shaped nuohan inferior yeast of slant preservation 37 DEG C of work
After changing 3～4h, in picking list colony inoculation to 5～10mL seed culture mediums, then in 37 DEG C, 160r/min
Lower shaken cultivation 18～24h obtains seed liquor A；Seed liquor A is coated height and oozes in solid medium, so
After in 37 DEG C of constant temperature culture 24～48h, then picking list colony inoculation is in 5～10mL seed culture mediums,
Then in 37 DEG C, shaken cultivation 18～24h obtains seed liquor B under 160r/min；Seed liquor B is coated
Height oozes in solid medium, and then in 37 DEG C of constant temperature culture 24～48h, then picking list colony inoculation is extremely
In 5～10mL seed culture mediums, then in 37 DEG C, shaken cultivation 18～24h obtains seed liquor under 160r/min
C；Seed liquor C is coated height and oozes in solid medium, then in 37 DEG C of constant temperature culture 24～48h.
Described fermentation seed is cultivated and is comprised the following steps: take the multiple-shaped nuohan inferior yeast list colony inoculation after domestication
In 5～10mL seed culture mediums, or by multiple-shaped nuohan inferior yeast after the domestication of slant preservation 37 DEG C of work
Changing 3～4h, after activation, picking list colony inoculation is in 5～10mL seed culture mediums, after inoculation in 37 DEG C,
Shaken cultivation 18～24h under 160～200r/min.
The composition of described seed culture medium includes: yeast extract 10g/L, glucose 20g/L and peptone
20g/L；The pH of seed culture medium is 6.0.
Described height oozes the composition of solid medium and includes: yeast extract 10g/L, glucose 500g/L, albumen
Peptone 20g/L and agar 15g/L；It is 6.0 that height oozes the pH of solid medium.
The composition of described fermentation medium includes: yeast extract 10g/L, glucose 400g/L, peptone
20g/L, magnesium sulfate 2.5g/L, potassium dihydrogen phosphate 3g/L, calcium chloride 0.5g/L and ammonium sulfate 3.75g/L；
The pH of fermentation medium is 6.0.
Beneficial effects of the present invention is embodied in:
D-R alcohol production method based on multiple-shaped nuohan inferior yeast of the present invention, according to yeast to height
Ooze the adaptation ability of environment and temperature stress stress on the impact of cellular metabolism, by comprehensive strain domestication (
Height ooze under the conditions of preculture) and regulation and control fermentation temperature technique, determine that multiple-shaped nuohan inferior yeast produces
The suitableeest production technology of D-arabitol, it is achieved improve the target of yeast production D-arabitol ability, through inspection
Survey, the present invention make dry cell weight and D-R alcohol yield respectively up to 5.26g/L and 114.92g/L,
Wherein D-R alcohol yield improves 30.50% than 37 DEG C of ferment at constant temperature, shows that the present invention can be used for
Improve the yield of multiple-shaped nuohan inferior yeast fermenting and producing D-R alcohol；And the method for the invention is prone to
Operation, cost is relatively low, be readily applied to produce D-R alcohol fermentation process in.
Detailed description of the invention
Below in conjunction with embodiment, the present invention is elaborated.
The present invention provides a kind of D-R alcohol production technology based on multiple-shaped nuohan inferior yeast: protected on inclined-plane
The multiple-shaped nuohan inferior yeast hidden oozes solid medium through height and carries out before strain fermentation after preculture, will be through preculture
Process the single bacterium colony obtained to be inoculated in fermentation medium after seed culture medium is cultivated and carry out temperature adjusting and send out
Ferment is cultivated, and first cultivates at a temperature of relatively low suitable yeast growth, after cell growth reaches stable phase；
Raise cultivation temperature, continue to cultivate the biosynthesis activating D-arabitol under stressful environmental；Finally will
Temperature drops to the suitableeest production temperature of D-arabitol, continues to cultivate, measure after harvesting Biomass and
D-arabitol yield.
Present disclosure is described in detail below:
(1) bacterial strain
Multiple-shaped nuohan inferior yeast (Hansenula polymorpha) DL-1 (source is ATCC No.26012),
It is preserved in Shaanxi Tech Univ's natural product Biotechnology Compose Experiment room.
(2) culture medium
Slant medium: yeast extract 10g/L, glucose 20g/L, peptone 20g/L, agar 15g/L,
PH=6.0.
Seed culture medium: yeast extract 10g/L, glucose 20g/L, peptone 20g/L, pH=6.0.
Fermentation medium: yeast extract 10g/L, glucose 400g/L, peptone 20g/L, magnesium sulfate 2.5g/L,
Potassium dihydrogen phosphate 3g/L, calcium chloride 0.5g/L, ammonium sulfate 3.75g/L, pH=6.0.
Height oozes solid medium: yeast extract 10g/L, glucose 500g/L, peptone 20g/L, agar
15g/L, pH=6.0.
The pH NaOH of culture medium or hydrochloric acid are adjusted.
(3) slant preservation
Multiple-shaped nuohan inferior yeast DL-1 is inoculated in slant medium, cultivates 18～24h in 37 DEG C and obtain inclined-plane
Seed, 4 DEG C of preservations are standby.
(4) strain domestication processes and preservation
By inclined-plane seed after 37 DEG C of activation 3h, one single colony inoculation of picking is trained to equipped with 5mL seed
Support in the test tube of base and carry out cultivating (condition of culture: 37 DEG C, shaken cultivation 24h under 160r/min) must plant
Sub-liquid A.Seed liquor A is coated the height in plate A ooze after solid medium in 37 DEG C of constant temperature culture
48h, then the most single bacterium colony of picking growth is forwarded in the test tube equipped with 5mL seed culture medium carry out
Cultivate (condition of culture: 37 DEG C, shaken cultivation 24h under 160r/min) seed liquor B.By seed liquor B
Coating the height in plate B to ooze after solid medium in 37 DEG C of constant temperature culture 48h, then picking grows relatively
Good single bacterium colony be forwarded in the test tube equipped with 5mL seed culture medium to carry out to cultivate (condition of culture: 37 DEG C,
Shaken cultivation 24h under 160r/min) obtain seed liquor C, high seed liquor C coated in plate C oozes
In 37 DEG C of constant temperature culture 48h after solid medium, domestication terminates.Then fermentation kind can be carried out by picking colony
Son is cultivated or strain after domestication is carried out slant preservation.
Strain inclined plane preservation after domestication: select bigger single bacterium colony in height oozes solid medium, in 37 DEG C
Cultivate after 18～24h standby in 4 DEG C of preservations.
(5) fermentation seed is cultivated
Single bacterium colony of strain after picking domestication, or by strain after the domestication of slant preservation 37 DEG C of activation
One single bacterium colony of picking after 3～4h, is seeded in the test tube equipped with 5mL seed culture medium carry out cultivating (training
Support condition: 160r/min, shaken cultivation 18～24h at 37 DEG C) fermentation seed culture fluid.
(6) temperature-variable fermentation is cultivated
Fermentation seed culture fluid is seeded to equipped with 100mL fermentation culture according to the inoculum concentration of 5% (v/v)
The triangular flask of base carries out three step alternating temperature regulation and control fermentation culture: first cultivate 18～24h at 30～38 DEG C,
Then cultivation temperature rises a height of 40～49 DEG C continue to cultivate 18～24 hours, finally cultivation temperature is dropped back to
28～38 DEG C are continued to cultivate 74～96h, and in fermentation culture, shaking speed remains 160～200r/min.
(7) detection and the outer yield of born of the same parents thereof of D-arabitol defines
Detection employing high performance liquid chromatography (HPLC) method of D-arabitol: chromatographic column SH1011；Flowing
Phase 0.01mol/L H2SO4；Flow velocity 0.8mL/min；Sample size 5 μ L；Column temperature 50 DEG C；Differential detects
Device (RID).
Content (the g)/fermentating liquid volume (L) of the outer yield=D-arabitol of the born of the same parents of D-arabitol (g/L)
(8) yeast cells dry weight (Dry Cell Wight, DCW)
Take the fermentation liquid of certain volume (L) centrifugal after with distilled water wash twice, the clean cell obtained
Dry to constant weight (g) at 85 DEG C, then calculate dry cell weight.
134 DEG C of ferment at constant temperature of embodiment
By one the single bacterium colony of picking after 37 DEG C of activation 3h of strain after the domestication of slant preservation, it is seeded to dress
There are in the test tube of 5mL seed culture medium and carry out cultivating (condition of culture: 160r/min, vibration training at 37 DEG C
Support 24h) obtain fermentation seed culture fluid.Fermentation seed culture fluid is connect according to the inoculum concentration of 5% (v/v)
Kind to equipped with the 500mL triangular flask of 100mL fermentation medium is carried out fermentation culture (160r/min, 34 DEG C
Lower shaken cultivation 144h) obtain fermentation liquid.In fermentation liquid, DCW is 5.09g/L, produces outside D-arabitol born of the same parents
Amount is 95.03g/L.
237 DEG C of ferment at constant temperature of embodiment
During fermentation culture, temperature is 37 DEG C, and other are same as in Example 1, and in fermentation liquid, DCW is
The outer yield of 5.28g/L, D-arabitol born of the same parents is 88.06g/L.
348 DEG C of ferment at constant temperature of embodiment
During fermentation culture, temperature is 48 DEG C, and other are same as in Example 1, and in fermentation liquid, DCW is
The outer yield of 3.21g/L, D-arabitol born of the same parents is 37.23g/L.
Embodiment 4 temperature-variable fermentation
By one the single bacterium colony of picking after 37 DEG C of activation 3h of strain after the domestication of slant preservation, it is seeded to dress
There are in the test tube of 5mL seed culture medium and carry out cultivating (condition of culture: 160r/min, vibration training at 37 DEG C
Support 24h) obtain fermentation seed culture fluid.Fermentation seed culture fluid is connect according to the inoculum concentration of 5% (v/v)
Plant to equipped with the 500mL triangular flask of 100mL fermentation medium carries out fermentation culture, fermentation culture mistake
Cheng Zhong, first cultivates 24h at 37 DEG C, then cultivation temperature rises a height of 44 DEG C and continues to cultivate 24 hours,
Cultivation temperature finally dropping back to 34 DEG C continue to cultivate 96h, shaking speed is always 160r/min, obtains fermentation
Liquid.In fermentation liquid, DCW is 5.31g/L；The outer yield of D-arabitol born of the same parents is 108.25g/L, relative to perseverance
Temperature fermentation (37 DEG C) improves 22.92%.
Embodiment 5 temperature-variable fermentation
By one the single bacterium colony of picking after 37 DEG C of activation 3h of strain after the domestication of slant preservation, it is seeded to dress
There are in the test tube of 5mL seed culture medium and carry out cultivating (condition of culture: 160r/min, vibration training at 37 DEG C
Support 24h) obtain fermentation seed culture fluid.Fermentation seed culture fluid is connect according to the inoculum concentration of 5% (v/v)
Plant to equipped with the 500mL triangular flask of 100mL fermentation medium carries out fermentation culture, fermentation culture mistake
Cheng Zhong, first cultivates 18h at 37 DEG C, then cultivation temperature rises a height of 48 DEG C and continues to cultivate 18 hours,
Cultivation temperature finally dropping back to 34 DEG C continue to cultivate 96h, shaking speed is always 160r/min, obtains fermentation
Liquid.In fermentation liquid, DCW is 5.26g/L；The outer yield of D-arabitol born of the same parents is 114.92g/L, relative to perseverance
Temperature fermentation (37 DEG C) improves 30.50%.
By the research to multiple-shaped nuohan inferior yeast DL-1 fermenting and producing D-R alcohol process, according to height
Ooze stressful environmental and temperature to growth of microbial cells and the impact of product catabolism process, grow in conjunction with cell
Optimum temperature, product generate optimum temperature and height ooze the stress conditions impact on cell, the present invention
A kind of D-R alcohol fermentation method for producing based on multiple-shaped nuohan inferior yeast, cell after fermentation ends are provided
Dry weight and D-R alcohol yield respectively up to 5.26g/L and 114.92g/L, wherein D-R
Alcohol yield improves 30.50% than 37 DEG C of ferment at constant temperature, shows that the method can be used for improving the inferior ferment of the multiform Chinese
The yield of female DL-1 fermenting and producing D-R alcohol.The method of the invention is easily operated, cost relatively
The fermentation process producing D-R alcohol low, that be readily applied in industrialized production, for D-arabitol
Industrial biological fermenting and producing provide practical basis.
Claims (7)
1. a D-R alcohol production method based on multiple-shaped nuohan inferior yeast, it is characterised in that: bag
Include following steps:
Carry out fermentation seed after being tamed under the conditions of height oozes by multiple-shaped nuohan inferior yeast and cultivate to obtain fermentation seed cultivation
Liquid, carries out fermentation culture after fermentation seed culture fluid is seeded to fermentation medium, the process of fermentation culture
For: first in 30～38 DEG C of shaken cultivation 18～24h, then cultivation temperature is risen a height of 40～49 DEG C of continuation
Shaken cultivation 18～24h, is then down to cultivation temperature 28～38 DEG C and continues shaken cultivation 74～96h, fermentation
In incubation, shaking speed remains 160～200r/min.
A kind of D-R alcohol producer based on multiple-shaped nuohan inferior yeast
Method, it is characterised in that: the source of described multiple-shaped nuohan inferior yeast is ATCC No.26012.
A kind of D-R alcohol producer based on multiple-shaped nuohan inferior yeast
Method, it is characterised in that: the acclimation method of described multiple-shaped nuohan inferior yeast is: by inferior for the multiform Chinese of slant preservation
Yeast is after 37 DEG C of activation 3～4h, in picking list colony inoculation to 5～10mL seed culture mediums, then
In 37 DEG C, shaken cultivation 18～24h obtains seed liquor A under 160r/min；Seed liquor A is coated height ooze
In solid medium, then in 37 DEG C of constant temperature culture 24～48h, then picking list colony inoculation is to 5～10mL
In seed culture medium, then in 37 DEG C, shaken cultivation 18～24h obtains seed liquor B under 160r/min；Will
Seed liquor B is coated height and is oozed in solid medium, then in 37 DEG C of constant temperature culture 24～48h, then chooses
Take single colony inoculation in 5～10mL seed culture mediums, then in 37 DEG C, shaken cultivation under 160r/min
18～24h obtain seed liquor C；Seed liquor C is coated height and oozes in solid medium, then in 37 DEG C of perseverances
Temperature cultivates 24～48h.
A kind of D-R alcohol producer based on multiple-shaped nuohan inferior yeast
Method, it is characterised in that: described fermentation seed is cultivated and is comprised the following steps: take the inferior ferment of the multiform Chinese after domestication
Female single colony inoculation is in 5～10mL seed culture mediums, or by inferior for the multiform Chinese after the domestication of slant preservation
Yeast activates 3～4h at 37 DEG C, and after activation, picking list colony inoculation is in 5～10mL seed culture mediums, connects
After Zhong under 37 DEG C, 160～200r/min shaken cultivation 18～24h.
5. raw according to a kind of D-R alcohol based on multiple-shaped nuohan inferior yeast described in claim 3 or 4
Product method, it is characterised in that: the composition of described seed culture medium includes: yeast extract 10g/L, glucose
20g/L and peptone 20g/L；The pH of seed culture medium is 6.0.
A kind of D-R alcohol producer based on multiple-shaped nuohan inferior yeast
Method, it is characterised in that: described height oozes the composition of solid medium and includes: yeast extract 10g/L, glucose
500g/L, peptone 20g/L and agar 15g/L；It is 6.0 that height oozes the pH of solid medium.
A kind of D-R alcohol producer based on multiple-shaped nuohan inferior yeast
Method, it is characterised in that: the composition of described fermentation medium includes: yeast extract 10g/L, glucose 400g/L,
Peptone 20g/L, magnesium sulfate 2.5g/L, potassium dihydrogen phosphate 3g/L, calcium chloride 0.5g/L and ammonium sulfate
3.75g/L；The pH of fermentation medium is 6.0.
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| CN104195059B (en) * | 2014-08-15 | 2016-08-24 | 陕西科技大学 | The method that multiple-shaped nuohan inferior yeast mutant strain and multiple-shaped nuohan inferior yeast produce D-R alcohol |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4271268A (en) * | 1977-09-12 | 1981-06-02 | Hoffmann-La Roche Inc. | Process for producing D-arabitol |
| CN103014103A (en) * | 2012-12-20 | 2013-04-03 | 陕西科技大学 | Method of producing glutathione through hansenula polymorpha fermentation |
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2014
- 2014-05-21 CN CN201410216345.0A patent/CN103981224B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4271268A (en) * | 1977-09-12 | 1981-06-02 | Hoffmann-La Roche Inc. | Process for producing D-arabitol |
| CN103014103A (en) * | 2012-12-20 | 2013-04-03 | 陕西科技大学 | Method of producing glutathione through hansenula polymorpha fermentation |
Non-Patent Citations (3)
| Title |
|---|
| Production of Arabitol from Glucose by Hansenula polymorpha;JAVIER ESCALANTE et al;《JOURNAL OF FERMENTATION AND BIOENGINEERING》;19901231;第70卷(第4期);228-231 * |
| 利用组合策略提高汉逊酵母发酵生产D-阿拉伯糖醇;钱卫东等;《安徽农业科学》;20140810;第42卷(第23期);7726-7728 * |
| 发酵法生产木糖醇中重要中间产物的发酵条件的研究;尤翠萍;《中国优秀硕士学位论文全文数据库工程科技I辑》;20110815(第08期);摘要,4-5,9-35 * |
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