CN102181374A

CN102181374A - High-permeability-resistant saccharomyces

Info

Publication number: CN102181374A
Application number: CN2011101059719A
Authority: CN
Inventors: 李政; 王玉; 张健飞
Original assignee: Tianjin Polytechnic University
Current assignee: Kou Shimei Tianjin Biotechnology Co Ltd
Priority date: 2011-04-27
Filing date: 2011-04-27
Publication date: 2011-09-14
Anticipated expiration: 2031-04-27
Also published as: CN102181374B

Abstract

The invention discloses high-permeability-resisting saccharomyces, particularly relating to a high-permeability-resistant brewing saccharomyces strain, belonging to the field of saccharomyces. The saccharomyces cerevisiae provided by the invention is preserved in the China General Microbiological Culture Collection Center (CGMCC) with the preservation number of CGMCC No.4429; and each performance index obtained by the experiment of the fermentation tank shows that the saccharmyces strain has the advantages of normal metabolism, strong ethanol generating capacity and low heteroacid content, thereby being an excellent high-permeability-resisting saccharomyces strain.

Description

One strain osmophilic yeast bacterium

Technical field:

The invention belongs to the yeast field, particularly anti-height oozes Wine brewing yeast strain.

Background technology:

In recent years, owing to have some special functions, the research of extreme microorganism causes that more and more people pay attention to.Wherein, osmophilic yeast is because it is in the widespread use of multiple industry such as the energy, food and medicine and particularly outstanding.At present it is mainly used and comprises following several aspect: (1) ethanol: ethanol is a kind of important solvent and fuel, utilizes the high concentration that yeast can improve substrate and product in the fermented liquid of oozing, and has multiple advantage such as reduce cost; (2) glycerine: when being in height and oozing in the environment, the yeast cell of most of kinds is beneficial to regulate the balance of osmotic pressure at intracellular accumulation glycerine; (3) erythritol: erythritol is a kind of sweet taste extender of appropriateness, and sugariness is 60%～70% of a sucrose, has the advantage of the carious tooth of preventing, suitable for patients with diabetes mellitus.Under height oozes condition, be beneficial to the accumulation of erythritol.In addition, height oozes yeast can appropriateness avoid pollution microbes aborning, reduces production risk.

Say that for microbiological industry it is very crucial how to obtain the good bacterial classification of a strain, so the isolation and selection work of yeast saccharomyces cerevisiae is most important, but will obtain good bacterial classification not a duck soup.Although adopt technique means such as genetically engineered can add one or more foreign genes in cell now, perhaps deleting certain gene is not difficult matter.But just it seems at present, obtains a bacterial strain with outstanding proterties and only depend on above simple genetic manipulation and be not easy, and delete certain gene or import foreign gene and may destroy metabolic balance in the yeast born of the same parents, and then the eubolism that influences cell is grown.In addition, for foodstuffs industry, the use of genetic engineering bacterium may have high safety hidden danger, and is forbidden in a lot of places.Therefore, traditional selection by mutation is still most important, the otherwise effective technique of most of industrial micro breedings.

The method that is used for microorganism mutation breeding at present has physics and chemomorphosis, wherein physical mutagenesis comprises physical methods such as ultraviolet ray, laser, X ray, gamma-rays, fast neutron, and chemomorphosis mainly comprises various alkylating agents (ethyl sulfate, nitrosoguanidine and ethylmethane sulfonate etc.).

Alkylating agent claims alkylating agent again, is the active class chemical substance of height that little alkyl can be transferred on other molecule.The general alkyl of introducing is connected on the atoms such as nitrogen, oxygen, carbon.The normal tool sudden change of alkylating agent source property is because it can change the Nucleotide in the thymus nucleic acid.Now having known has several different chemotherapeutic agents to belong to alkylating agent.They have one or two alkyl, divide single function of another name or difunctional alkylating agent, contained alkyl can play alkanisation with nucleophilic group in DNA, the RNA of cell or the protein, often can form cross bracing or cause depurination, make the DNA splitting of chain, when duplicating, can make the base pairing error code again next time, cause the infringement of dna structure and function, can cause necrocytosis when serious.Belong to cell cycle nonspecific agent (CCNSA).Alkylating agent commonly used has alkene, alkyl halide, sulfuric acid alkane ester etc.

And these alkylating agents are in traditional mutagenesis operation, and selection by mutation is effective, the simple advantage of operational condition because it has, and is widely used.

Summary of the invention:

Technical problem to be solved by this invention provides the new anti-height of a strain and oozes Wine brewing yeast strain and the application aspect ethanol fermentation thereof.

Yeast saccharomyces cerevisiae provided by the present invention (Saccharomyces cerevisiae), this bacterial strain is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, deposit number is CGMCC No.4429, preservation address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode 100101.Preservation date on December 08th, 2010.

These bacterial strain characteristics are as follows:

Examine under a microscope, the cell of this bacterial strain is avette, an end budding, and size is about 1 * 5 μ m; On solid medium, this bacterium bacterium colony is an oyster white, and smooth surface is moistening, thickness, and the edge is more neat and medium bigger than normal.Compare with original bacterium, this mutagenic strain is significantly less than starting strain on form.

Starting strain yeast saccharomyces cerevisiae CICC31481 purchases in Chinese industrial microbial strains preservation administrative center.

Yeast strain of the present invention adopts following flow process to carry out seed selection:

The original bacterial classification that sets out → test tube activation → ethyl sulfate (DES) mutagenesis → height oozes plate screening → nitrosoguanidine (NTG) mutagenesis screening → height and oozes dull and stereotyped primary dcreening operation → shake bottle multiple sieve → mitotic stability test → 7L fermentor tank test

The present invention adopts DES that starting strain is carried out mutagenesis earlier, ooze flat board (150g/L KCl) substratum primary dcreening operation by the wort height after the mutagenesis, then the bacterial strain that seed selection is come out is proceeded NTG mutagenesis, ooze flat board (200g/LKCl) substratum primary dcreening operation by the wort height, adopt the 250mL triangular flask to sieve again then, the yeast strain that seed selection is good, do the experiment of going down to posterity then, estimate its genetic stability, and measure metabolite content in the fermented liquid with liquid chromatography, gas chromatograph, gas chromatography mass spectrometry, adopt the 7L fermentor tank Evaluation on effect that experimentizes at last.

Bacterial strain CGMCC No.4429 genetic stability is the result show: through continuous passage ten times, every performance index are all more stable, and heredity is better, and proterties is not replied, therefore the purpose bacterial strain that bacterial strain CGMCC No.4429 is obtained as seed selection.

Purpose bacterial strain CGMCC No.4429 is done the experiment of 7L fermentor tank, and the result shows: compare with starting strain, CGMCCNo.4429 initial glucose tolerance concentration can reach 300g/L, compares with original bacterium and has improved 50%; After the fermentation ends, residual sugar is that 0.5g/L, glycerol content are 1g/L, compares with original bacterium and has improved 10%; Acetic acid content is that 0.8g/L, lactic acid content are 0.4g/L, and the original bacterium of fundamental sum is suitable; Alcohol concn is 175g/L, compares with original bacterium and has improved 108%.

Beneficial effect:

1) DES and NTG mutagenesis coupling technique seed selection Wine brewing yeast strain are adopted in this research, and seed selection has obtained strain excellent CGMCC No.4429.This mutant strain can tolerate 200g/L KCl.The glucose tolerance is 300g/L, compares with original bacterium and has improved 50%.This bacterial strain genetic stability is good, and in continuous ten processes that go down to posterity, proterties is not replied, and every performance index are all normal.

2) every performance index of fermentor tank experiment tracking show that this bacterial strain metabolism is normal, and the producing and ethanol ability is strong, and heteroacid content is low, is that the good anti-height of a strain oozes Wine brewing yeast strain.

Embodiment:

The following examples can make those skilled in the art more fully understand the present invention, but do not limit the present invention in any way.

Give an example 1:

Detailed process is as follows:

1.DES mutagenic and breeding

1) at S. cervisiae CICC31481 one ring of getting on the super clean bench on the test tube slant, insert and be equipped with in the 250mL triangular flask of 50mL malt extract medium, 200rpm cultivates about 10h for 30 ℃, makes thalline be in logarithmic growth in earlier stage.

2) get 5mL bacterium liquid, the centrifugal 10min of 5000rpm collects thalline, with physiological saline washing 2 times.

3) be diluted to 10 with the pH7.0 phosphoric acid buffer ⁷Individual/the mL bacteria suspension.

4) potassium phosphate buffer, 8mL bacteria suspension, 0.4mL DES of getting 32mL pH7.0 put into the 150mL triangular flask thorough mixing of rotor in advance, and making the DES ultimate density is 1% (v/v).

5) 150rpm reaction 30min in 30 ℃ of shaking tables gets the 1mL mixed solution, adds 0.5mL 25%Na ₂S ₂O ₃The solution stopped reaction.

6) dilution is coated in the wort screening culture medium plate that contains 150g/L KCl.At the bacterial strain of 30 ℃ of cultivations picking colony maximum after 2～3 days, label is the D bacterium.

2. nitrosoguanidine mutagenesis

1) at S. cervisiae one ring of getting on the super clean bench on the test tube slant, insert and be equipped with in the 250mL triangular flask of 50mL malt extract medium, 200rpm cultivates about 10h for 30 ℃, makes thalline be in logarithmic growth in earlier stage.

2) get the centrifugal 10min of 5mL bacterium liquid 5000rpm and collect thalline, with physiological saline washing 2 times.

3) be diluted to 10 with the pH6.0 phosphoric acid buffer ⁷Individual/the mL bacteria suspension.

4) get the 10mL bacteria suspension and be transferred in the 100mL triangular flask, add the NTG of 10mg, being mixed with final concentration is the NTG solution of 10mg/mL, and adds 4-5 and drip acetone, is beneficial to the NTG dissolving.

5) at 30 ℃ of following 200rpm oscillatory reaction 30min, the centrifugal 10min of 5000rpm collects thalline, with the stroke-physiological saline solution washing for several times, and stopped reaction.

6) suitably dilution is coated with, and gets last dilution bacterium liquid 0.2mL, coats in the wort screening culture medium plate that contains 200g/L KCl.30 ℃ cultivate 2～3 days after 60 of picking colonies.

3. shake the multiple sieve of bottle

1) at S. cervisiae one ring of getting respectively on the super clean bench on each test tube slant, insert and to be equipped with in the 250mL triangular flask of 50mL malt extract medium, 200rpm cultivates about 12h for 30 ℃, makes thalline be in logarithmic growth mid-term.

2) get 5mL bacterium liquid, insert and the 50mL height to be housed to ooze in the 250mL triangular flask in the malt extract medium (glucose concn is 300g/L), 200rpm cultivated 3-4 days for 30 ℃, detected glucose concn and alcohol concn every day and changed.After the fermentation ends, relatively the glucose of 60 strain bacterial classifications and ethanol wear rate, final remaining sugar concentration and alcohol concn, glucose are to ethanol conversion and heteroacid content.

3) selection glucose and ethanol wear rate are soon, final remaining sugar concentration is low and alcohol concn is high, glucose is final bacterial classification to ethanol conversion height and the poor bacterial classification of heteroacid, called after DN bacterium.

4. genetic stability test

The DN bacterium is gone down to posterity for continuous ten times on the inclined-plane, and detect fermentation situation after at every turn going down to posterity with the method for shaking the multiple sieve of bottle.Experiment finds, goes down to posterity for continuous ten times on the inclined-plane, and this bacterial classification proterties does not have considerable change, and every performance index are all normal, illustrate that the genetic stability of this bacterial classification is stronger.

5.7L fermentor tank test

1) get S. cervisiae one ring on the inclined-plane, insert and be equipped with in the 250mL triangular flask of 50mL malt extract medium, 200rpm cultivates about 12h for 30 ℃, makes thalline be in logarithmic growth mid-term.

2) the bacterial classification access of logarithmic phase is equipped with in the 7L fermentor tank of 4L fermented liquid.Inoculum size is 10%, 30 ℃ of following 100rpm, logarithm dissolved oxygen in early stage control 10% (ventilation 0.5L/min), and the later stage anaerobism was cultivated 3-4 days.

3) after the fermentation ends, residual sugar is that 0.5g/L, glycerol content are 1g/L, compares with original bacterium and has improved 10%; Acetic acid content is that 0.8g/L, lactic acid content are 0.4g/L, and the original bacterium of fundamental sum is suitable; Alcohol concn is 175g/L, compares with original bacterium and has improved 108%.

Claims

1. a strain osmophilic yeast bacterium (Saccbaromyces cerevisiae), deposit number is CGMCC No.4429.