CN102533591A - High temperature resisting and high-glucose resisting lactic acid bacteria - Google Patents
High temperature resisting and high-glucose resisting lactic acid bacteria Download PDFInfo
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- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 title claims abstract description 28
- 239000008103 glucose Substances 0.000 title claims abstract description 23
- 235000014655 lactic acid Nutrition 0.000 title claims abstract description 14
- 239000004310 lactic acid Substances 0.000 title claims abstract description 10
- 241000894006 Bacteria Species 0.000 title abstract description 36
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims abstract description 19
- 241000218588 Lactobacillus rhamnosus Species 0.000 claims abstract description 17
- 238000005303 weighing Methods 0.000 claims 2
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- 238000000855 fermentation Methods 0.000 abstract description 6
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- PLUBXMRUUVWRLT-UHFFFAOYSA-N Ethyl methanesulfonate Chemical compound CCOS(C)(=O)=O PLUBXMRUUVWRLT-UHFFFAOYSA-N 0.000 description 1
- 244000068988 Glycine max Species 0.000 description 1
- 235000010469 Glycine max Nutrition 0.000 description 1
- NVNLLIYOARQCIX-MSHCCFNRSA-N Nisin Chemical compound N1C(=O)[C@@H](CC(C)C)NC(=O)C(=C)NC(=O)[C@@H]([C@H](C)CC)NC(=O)[C@@H](NC(=O)C(=C/C)/NC(=O)[C@H](N)[C@H](C)CC)CSC[C@@H]1C(=O)N[C@@H]1C(=O)N2CCC[C@@H]2C(=O)NCC(=O)N[C@@H](C(=O)N[C@H](CCCCN)C(=O)N[C@@H]2C(NCC(=O)N[C@H](C)C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCSC)C(=O)NCC(=O)N[C@H](CS[C@@H]2C)C(=O)N[C@H](CC(N)=O)C(=O)N[C@H](CCSC)C(=O)N[C@H](CCCCN)C(=O)N[C@@H]2C(N[C@H](C)C(=O)N[C@@H]3C(=O)N[C@@H](C(N[C@H](CC=4NC=NC=4)C(=O)N[C@H](CS[C@@H]3C)C(=O)N[C@H](CO)C(=O)N[C@H]([C@H](C)CC)C(=O)N[C@H](CC=3NC=NC=3)C(=O)N[C@H](C(C)C)C(=O)NC(=C)C(=O)N[C@H](CCCCN)C(O)=O)=O)CS[C@@H]2C)=O)=O)CS[C@@H]1C NVNLLIYOARQCIX-MSHCCFNRSA-N 0.000 description 1
- 108010053775 Nisin Proteins 0.000 description 1
- 241000237502 Ostreidae Species 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
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- WRUGWIBCXHJTDG-UHFFFAOYSA-L magnesium sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Mg+2].[O-]S([O-])(=O)=O WRUGWIBCXHJTDG-UHFFFAOYSA-L 0.000 description 1
- ISPYRSDWRDQNSW-UHFFFAOYSA-L manganese(II) sulfate monohydrate Chemical compound O.[Mn+2].[O-]S([O-])(=O)=O ISPYRSDWRDQNSW-UHFFFAOYSA-L 0.000 description 1
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Abstract
The invention discloses high temperature resisting and high-glucose resisting lactic acid bacteria, and belongs to the field of lactic acid bacteria, in particular to high temperature resisting and high-glucose resisting strains. The lactic acid bacteria provided by the invention is Lactobacillus rhamnosus which is stored in China General Microbiological Culture Collection Center with a collection number of CGMCC No. 4430. The glucose tolerance concentration of the strain CGMCC No. 4430 reaches 270g/L which is improved by 95% compared to the glucose tolerance concentration of the primary strain. After fermentation, the content of lactic acid is 50g/L which is increased by 158% compared to the concentration of the primary strain. The performance indicators followed in fermentation tank experiment show that the strain which is normal in metabolism and has strong L-lactic acid-producing capacity and low content of heteroacid is a high temperature resisting and high-glucose resisting Lactobacillus rhamnosus strain.
Description
Technical field:
The invention belongs to the probiotic lactobacillus field, the lactobacillus strains of particularly high temperature resistant, anti-high sugar.
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, high temperature resistant, anti-height oozes milk-acid bacteria because it is in the widespread use of multiple industry such as food, medicine and chemical industry and particularly outstanding.It is mainly used and comprises following several aspect at present: (1) lactic acid: lactic acid is a kind of important food and industrial chemicals, and can prepare degradable high polymer material-POLYACTIC ACID, thereby receives much concern in recent years.(2) biological preservative: nisin is a kind of biological preservative that was widely used in food and drink in recent years, compares with Chemical Preservative, and it is harmless, and has wider antimicrobial spectrum.(3) food: yogurt, cheese, pickles, fermented soya bean etc. all are a kind of functional foodstuffs of extensively liking of each country in the world, and they all are fermented bacterium with the milk-acid bacteria.Blend under the pyritous condition at height, be beneficial to the concentration that improves substrate and product.In addition, can reduce pollution microbes aborning, reduce 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 milk-acid bacteria is most important, but will obtain good bacterial classification not a duck soup.Although adopt technique means such as genetically engineered can add foreign gene in cell now, perhaps delete certain gene.But just technology it seems at present; Obtaining a bacterial strain with good character only depends on above simple genetic manipulation and is not easy; And delete certain gene or import foreign gene and may destroy the metabolic balance in the lactic acid mycetocyte, and then influence the homergy growth of cell.In addition, for foodstuffs industry, the use of genetic engineering bacterium possibly have high safety hidden danger, is forbidden in a lot of countries.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 is claimed alkylating agent again, is can little alkyl be transferred to the active one type of chemical substance of height 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.Known has several kinds of different chemotherapeutic agents to belong to alkylating agent at present.They have one or two alkyl, divide single function of another name or difunctional alkylating agent, and contained alkyl can play alkanisation with nucleophilic group in DNA, RNA or the protein of cell; 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 new lactobacterium strain of a strain and the application aspect lactobacillus ferment thereof.
Probiotic lactobacillus provided by the present invention is lactobacillus rhamnosus (Lactobacillus rhamnosus); This bacterial strain is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center; Protect a surname and be numbered CGMCC No.4430; 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 following: examine under a microscope, this bacterial strain is shaft-like, and width is less than 1 μ m, and 2 to 3 bacillus are easy to be linked to be and link together; On solid medium, this bacterium bacterium colony is an oyster white, and smooth surface is moistening, thickness, and the edge is more neat.Compare with original bacterium, this mutagenic strain is significantly less than starting strain on form.
Starting strain lactobacillus rhamnosus CGMCC No.1.2134 purchases in China Committee for Culture Collection of Microorganisms common micro-organisms center.
Lactobacillus rhamnosus of the present invention adopts following flow process to carry out seed selection:
A bottle multiple sieve → mitotic stability test → 5L fermentor tank test is screened → shaken to the original bacterial classification that sets out → test tube activation → high temperature acclimation → ethyl sulfate (DES) mutagenesis → high sugared plate screening → nitrosoguanidine (NTG) mutagenesis screening → high temperature bacterium
The righttest leavening temperature of original strain that the present invention adopted is 37 ℃, in order to improve its resistant to elevated temperatures character, at first adopts and progressively improves method of temperature and tame.Can be till 45 ℃ of dull and stereotyped can growths up to it.Adopt DES to carry out further mutagenesis then to obtaining the high temperature bacterium; Carry out primary dcreening operation through the high sugar of culture medium A dull and stereotyped (250g/L glucose) after the mutagenesis, the bacterial strain that then seed selection is come out is proceeded NTG mutagenesis, carries out high temperature bacterium (tolerating 55 ℃) primary dcreening operation through the high sugar of culture medium A dull and stereotyped (250g/L glucose); Adopt the 250mL triangular flask to sieve again then; The lactobacillus strains that seed selection is good is done the experiment of going down to posterity then, estimates its genetic stability; And measure the metabolite content in the fermented liquid with liquid chromatography, gas chromatography mass spectrometry, adopt the 5L fermentor tank Evaluation on effect that experimentizes at last.
Bacterial strain CGMCC No.4430 genetic stability is the result show: through continuous passage ten times, each item performance index are all more stable, and heredity is better, and proterties is not replied, the purpose bacterial strain that therefore obtains bacterial strain CGMCC No.4430 as seed selection.
Purpose bacterial strain CGMCC No.4430 is done the experiment of 5L lactobacillus ferment jar, and the result shows: compare with starting strain, CGMCC No.4430 glucose tolerance concentration can reach 270g/L, compares with original bacterium and has improved 95%; After the fermentation ends, lactic acid content is 60g/L, compares with original bacterium and has improved 158%.
Beneficial effect:
1) DES and NTG mutagenesis coupling technique seed selection lactobacillus rhamnosus are adopted in this research, and seed selection has obtained strain excellent CGMCC No.4430.This mutant strain can be at 55 ℃ of following well-growns, and fermention medium does not need sterilization.The glucose tolerance is 270g/L, compares with original bacterium and has improved 95%.This bacterial strain genetic stability is good, and in continuous ten processes that go down to posterity, proterties is not replied, and each item performance index are all normal.
2) each item performance index of fermentor tank experiment tracking show that this bacterial strain metabolism is normal, and product L-lactic acid ability is strong, and heteroacid content is low, is the good sugared lactobacillus rhamnosus strain of high temperature resistant, anti-height of a strain.
Embodiment:
Following embodiment can make those skilled in the art more fully understand the present invention, but does not limit the present invention in any way.
Give an example 1:
Detailed process is following:
1. high temperature acclimation
1) at lactobacillus rhamnosus CGMCC No.1.2134 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 culture medium A (no agar), 200rpm cultivates about 12h for 37 ℃, makes thalline be in logarithmic growth in earlier stage.
Culture medium A: (casein peptone 10.0g, Carnis Bovis seu Bubali cream 10.0g, yeast powder 5.0g, glucose 5.0g; Sodium acetate 5.0g, Hydrocerol A diamines 2.0g, Tween 80 1.0g, potassium hydrogenphosphate 2.0g; MAGNESIUM SULPHATE HEPTAHYDRATE 99.5 0.2g, manganese sulfate monohydrate 0.05g, lime carbonate 20.0g; Agar 15.0g, zero(ppm) water 1.0L, pH6.8).
2) get 5mL bacterium liquid, the centrifugal 10min of 5000rpm collects thalline, with saline water washing 2 times.
3) be diluted to 10 with saline water
7Individual/the mL bacteria suspension.
4) dilution is coated in the plate that contains culture medium A.At the bacterial strain of 39 ℃ of cultivations picking colony maximum after 2～3 days, label is the H1 bacterium.
5) repeat top method, with screening to such an extent that the bacterium dilution is coated in the plate that contains culture medium A.At the bacterial strain of 41 ℃ of cultivations picking colony maximum after 2～3 days, label is the H2 bacterium.
6) repeat above operation, culture temperature improves 2 ℃ at every turn, can be until filtering out at the bacterial strain of 45 ℃ of growths, and label is the H bacterium.
2.DES mutagenic and breeding
1) at lactobacillus rhamnosus H 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 culture medium A (no agar) substratum, 200rpm cultivates about 12h for 45 ℃, makes thalline be in logarithmic growth in earlier stage.
2) get 5mL bacterium liquid, the centrifugal 10min of 5000rpm collects thalline, with saline water 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 culture medium A screening culture medium plate that contains 250g/L glucose.At the bacterial strain of 45 ℃ of cultivations picking colony maximum after 2～3 days, label is the HG bacterium.
3. nitrosoguanidine mutagenesis
1) at lactobacillus rhamnosus HG one ring of getting on the super clean bench on the test tube slant; Access is equipped with in the 250mL triangular flask of 50mL culture medium A (no agar) substratum (glucose concn is 250g/L); 200rpm cultivates about 12h for 45 ℃, makes thalline be in logarithmic growth in earlier stage.
2) get the centrifugal 10min of 5mL bacterium liquid 5000rpm and collect thalline, with saline water 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 SPSS washing for several times, and stopped reaction.
6) suitably dilution is coated with, and gets last dilution bacterium liquid 0.2mL, coats in the culture medium A screening culture medium plate that contains 250g/L glucose.55 ℃ cultivate 2～3 days after bigger 100 of picking colony.
4. shake the multiple sieve of bottle
1) at lactobacillus rhamnosus one ring of getting respectively on the super clean bench on each test tube slant; Access is equipped with in the 250mL triangular flask of 50mL culture medium A (no agar) substratum (glucose concn is 250g/L); 200rpm cultivates about 15h for 30 ℃, makes thalline be in logarithmic growth mid-term.
2) get 5mL bacterium liquid, insert and be equipped with in the 250mL triangular flask among the 50mL high glucose medium A (no agar) (glucose concn is 250g/L), 200rpm cultivated 3-4 days for 30 ℃, detected glucose concn and L-lactic acid concn every day and changed.After the fermentation ends, relatively the glucose consumption speed of 100 strain bacterial classifications and L-lactic acid generation speed, glucose are to the transformation efficiency and the heteroacid content of L-lactic acid.
3) selection glucose consumption speed soon, final remaining sugar concentration is low and the L-lactic acid concn high, glucose is final bacterial classification to the transformation efficiency height and the poor bacterial classification of heteroacid of L-lactic acid, called after HGN bacterium.
5. genetic stability test
The HGN bacterium is gone down to posterity for continuous ten times on the inclined-plane, and detect the fermentation situation after at every turn going down to posterity with a method of shaking the multiple sieve of bottle.Experiment finds, on the inclined-plane, goes down to posterity for continuous ten times, and this bacterial classification proterties does not have considerable change, and each item performance index are all normal, explain that the genetic stability of this bacterial classification is stronger.
6.5L fermentor tank test
1) get lactobacillus rhamnosus one ring on the inclined-plane, insert and be equipped with in the 250mL triangular flask of 50mL culture medium A (no agar) (glucose concn is 150g/L) substratum, 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 5L fermentor tank of 3L 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 90g/L, L-lactic acid content are 50g/L, compare with original bacterium and have improved 158%.
Claims (3)
1. the lactobacillus rhamnosus (Lactobacillus rhamnosus) of high temperature resistant, the anti-high sugar of a strain, deposit number is CGMCCNo.4430.
2. as weighing 1 said high temperature resistant, anti-high sugared lactobacillus rhamnosus CGMCC No.4430 at 55 ℃ of following well-growns, tolerance 250g/L glucose.
3. as weighing the application of lactobacillus rhamnosus CGMCC No.4430 in lactic acid-producing of 1 said high temperature resistant, anti-high sugar.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201110419568 CN102533591B (en) | 2011-12-15 | 2011-12-15 | High temperature resisting and high-glucose resisting lactic acid bacteria |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201110419568 CN102533591B (en) | 2011-12-15 | 2011-12-15 | High temperature resisting and high-glucose resisting lactic acid bacteria |
Publications (2)
| Publication Number | Publication Date |
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| CN102533591A true CN102533591A (en) | 2012-07-04 |
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Cited By (5)
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| CN104673691A (en) * | 2013-11-29 | 2015-06-03 | 田岗 | Novel lactobacillus plantarum for high-yield production of lactic acid by efficiently utilizing biomass material |
| CN106858230A (en) * | 2015-12-14 | 2017-06-20 | 湖南斯奇生物制药有限公司 | A kind of carrot fermented beverage and preparation method thereof |
| EP3237600A4 (en) * | 2014-10-01 | 2018-08-01 | Triphase Pharmaceuticals Pvt. Ltd. | Thermo-stable strains, products and methods thereof |
| CN109517754A (en) * | 2018-11-20 | 2019-03-26 | 上海交通大学 | A method of high temperature bacterial strain is isolated and purified using common biochemical equipment |
| CN115011498A (en) * | 2021-03-05 | 2022-09-06 | 丰益(上海)生物技术研发中心有限公司 | High-temperature-resistant and high-sugar-resistant pediococcus acidilactici |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104673691A (en) * | 2013-11-29 | 2015-06-03 | 田岗 | Novel lactobacillus plantarum for high-yield production of lactic acid by efficiently utilizing biomass material |
| EP3237600A4 (en) * | 2014-10-01 | 2018-08-01 | Triphase Pharmaceuticals Pvt. Ltd. | Thermo-stable strains, products and methods thereof |
| CN106858230A (en) * | 2015-12-14 | 2017-06-20 | 湖南斯奇生物制药有限公司 | A kind of carrot fermented beverage and preparation method thereof |
| CN109517754A (en) * | 2018-11-20 | 2019-03-26 | 上海交通大学 | A method of high temperature bacterial strain is isolated and purified using common biochemical equipment |
| CN115011498A (en) * | 2021-03-05 | 2022-09-06 | 丰益(上海)生物技术研发中心有限公司 | High-temperature-resistant and high-sugar-resistant pediococcus acidilactici |
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