CN102533591B - 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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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 Bacterium lacticum field, particularly the lactobacillus strains of high temperature resistant, anti-high sugar.
Background technology:
In recent years, owing to having some special functions, the research of extreme microorganism causes that more and more people pay attention to.Wherein, high temperature resistant, anti-height ooze milk-acid bacteria due to it in the widespread use of the 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-poly(lactic acid), thereby receives much concern in recent years.(2) biological preservative: nisin is a kind of biological preservative that was widely used in recent years food and drink, compares with Chemical Preservative, and it is harmless, and has wider antimicrobial spectrum.(3) food: yogurt, cheese, pickles, fermented soya bean etc. are all a kind of functional foodstuffs of extensively liking of every country in the world, and they are all take milk-acid bacteria as fermented bacterium.Blend at height under the condition of high temperature, be beneficial to the concentration that improves substrate and product.In addition, can reduce pollution microbes aborning, reduce production risk.
Say for microbiological industry how to obtain the good bacterial classification of a strain very crucial, so the isolation and selection work of milk-acid bacteria is most important, but will obtains good bacterial classification not a duck soup.Although adopt now the technique means such as genetically engineered can add foreign gene in cell, perhaps delete certain gene.But just technology be 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 interior metabolic balance of lactic acid mycetocyte, and then affect the eubolism growth of cell.In addition, for foodstuffs industry, the use of genetic engineering bacterium may have high safety hidden danger, is forbidden in a lot of countries.Therefore, traditional selection by mutation is still that the most industry Microbial Breeding is most important, the most effective technology.
The method that is used at present microorganism mutation breeding has physics and chemistry mutagenesis, wherein physical mutagenesis comprises the 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 again alkylating agent, 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 thymus nucleic acid.Now having known has several different chemotherapeutic agents to belong to alkylating agent.They have one or two alkyl, divide the single function of another name or difunctional alkylating agent, contained alkyl can play alkanisation with nucleophilic group in DNA, the RNA of cell or protein, often can form cross bracing or cause depurination, make the DNA splitting of chain, when copying, 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 is to provide the new lactobacterium strain of a strain and the application aspect lactic fermentation thereof.
Bacterium lacticum provided by the present invention is lactobacillus rhamnosus (Lactobacillus rhamnosus), this bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's 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 as follows: 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 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 is purchased from China Committee for Culture Collection of Microorganisms's common micro-organisms center.
Lactobacillus rhamnosus of the present invention adopts following flow process to carry out seed selection:
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 screening → shaking flask is sieved → test of mitotic stability test → 5L fermentor tank again
The suitableeest leavening temperature of original strain of the present invention is 37 ℃, in order to improve its resistant to elevated temperatures character, at first adopts the method that progressively improves temperature to tame.Until they can be 45 ℃ of dull and stereotyped can growths.then adopt DES to carry out further mutagenesis to obtaining the high temperature bacterium, carry out primary dcreening operation by the high sugar of culture medium A dull and stereotyped (250g/L glucose) after mutagenesis, then seed selection bacterial strain is out proceeded NTG mutagenesis, carry out high temperature bacterium (tolerating 55 ℃) primary dcreening operation by the high sugar of culture medium A dull and stereotyped (250g/L glucose), then adopt the 250mL triangular flask to sieve again, the lactobacillus strains that seed selection is good, then do the experiment of going down to posterity, estimate its genetic stability, and use liquid chromatography, gas chromatography mass spectrometry is measured the metabolite content in fermented liquid, adopt at last the 5L fermentor tank to carry out the evaluation of experiment effect.
Bacterial strain CGMCC No.4430 genetic stability result shows: through continuous passage ten times, property indices is all more stable, and heredity is better, and proterties is not replied, the purpose bacterial strain that therefore bacterial strain CGMCC No.4430 is obtained as seed selection.
Purpose bacterial strain CGMCC No.4430 is done the experiment of 5L lactic acid fermentation tank, and result shows: compare with starting strain, CGMCC No.4430 glucose-tolerant concentration can reach 270g/L, compares with original bacterium and has improved 95%; After 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 lower well-growns, and fermention medium does not need sterilization.The glucose-tolerant ability 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 property indices is all normal.
2) property indices of fermentor tank follow-up experiment shows, this bacterial strain Metabolism of Normal, and product Pfansteihl 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:
The following examples can make the present invention of those skilled in the art's comprehend, but do not limit the present invention in any way.
Give an example 1:
Detailed process is as follows:
1. high temperature acclimation
1) at lactobacillus rhamnosus CGMCC No.1.2134 one ring of getting on super clean bench on the test tube slant, access is equipped with in the 250mL triangular flask of 50mL culture medium A (without agar), and 200rpm cultivates the 12h left and right for 37 ℃, makes thalline be in logarithmic growth in earlier stage.
Culture medium A: (casein peptone 10.0g, extractum carnis 10.0g, yeast powder 5.0g, glucose 5.0g, sodium acetate 5.0g, citric acid diamines 2.0g, Tween 80 1.0g, dipotassium hydrogen phosphate 2.0g, magnesium sulfate heptahydrate 0.2g, manganese sulfate monohydrate 0.05g, calcium carbonate 20.0g, agar 15.0g, distilled water 1.0L, pH6.8).
2) get 5mL bacterium liquid, the centrifugal 10min of 5000rpm collects thalline, with physiological saline washing 2 times.
3) become 10 with normal saline dilution
7Individual/mL bacteria suspension.
4) dilution spread is 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 above method, with screen the bacterium dilution spread 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 at the bacterial strain of 45 ℃ of growths until filter out, and label is the H bacterium.
2.DES mutagenic and breeding
1) at lactobacillus rhamnosus H one ring of getting on super clean bench on the test tube slant, access is equipped with in the 250mL triangular flask of 50mL culture medium A (without agar) substratum, and 200rpm cultivates the 12h left and right for 45 ℃, 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/mL bacteria suspension.
4) potassium phosphate buffer, 8mL bacteria suspension, 0.4mL DES of getting 32mL pH7.0 fully mixes at the 150mL triangular flask of putting in advance rotor, and making the DES ultimate density is 1% (v/v).
5) 150rpm reaction 30min in 30 ℃ of shaking tables, get the 1mL mixed solution, adds 0.5mL 25%Na
2S
2O
3The solution stopped reaction.
6) dilution spread is 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 super clean bench on the test tube slant, access is equipped with in the 250mL triangular flask of 50mL culture medium A (without agar) substratum (glucose concn is 250g/L), 200rpm cultivates the 12h left and right 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 physiological saline washing 2 times.
3) be diluted to 10 with the pH6.0 phosphoric acid buffer
7Individual/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 to drip acetone, is beneficial to the NTG dissolving.
5) at 30 ℃ of lower 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 culture medium A screening culture medium plate that contains 250g/L glucose.55 ℃ cultivate 2～3 days after larger 100 of picking colony.
4. shaking flask is sieved again
1) at lactobacillus rhamnosus one ring of getting respectively on super clean bench on each test tube slant, access is equipped with in the 250mL triangular flask of 50mL culture medium A (without agar) substratum (glucose concn is 250g/L), 200rpm cultivates the 15h left and right for 30 ℃, makes thalline be in logarithmic growth mid-term.
2) get 5mL bacterium liquid, access in the 250mL triangular flask that is equipped with in 50mL high glucose medium A (without agar) (glucose concn is 250g/L), 200rpm cultivated 3-4 days, and detected glucose concn and Pfansteihl change in concentration every day for 30 ℃.After fermentation ends, relatively the glucose consumption speed of 100 strain bacterial classifications and Pfansteihl produce speed, glucose to transformation efficiency and the heteroacid content of Pfansteihl.
3) select that fast, the final remaining sugar concentration of glucose consumption speed is low and Pfansteihl concentration is high, the high and poor bacterial classification of heteroacid is final bacterial classification to glucose to the transformation efficiency of Pfansteihl, 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 fermentation situation after at every turn going down to posterity with the method that shaking flask is sieved again.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 property indices is all normal, illustrates 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, access is equipped with in the 250mL triangular flask of 50mL culture medium A (without agar) (glucose concn is 150g/L) substratum, and 200rpm cultivates the 12h left and right 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 lower 100rpm, and logarithm dissolved oxygen in early stage is controlled 10% (ventilation 0.5L/min), and the later stage anaerobism was cultivated 3-4 days.
3) after fermentation ends, residual sugar 90g/L, Pfansteihl content are 50g/L, compare with original bacterium and have improved 158%.
Claims (2)
1. the lactobacillus rhamnosus (Lactobacillus rhamnosus) of high temperature resistant, the anti-high sugar of a strain, deposit number is CGMCCNo.4430.
2. the lactobacillus rhamnosus CGMCC No.4430 of high temperature resistant, anti-high sugar as claimed in claim 1, is characterized in that, this bacterial strain is at 55 ℃ of lower well-growns, tolerance 250g/L glucose.
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| CN 201110419568 CN102533591B (en) | 2011-12-15 | 2011-12-15 | High temperature resisting and high-glucose resisting lactic acid bacteria |
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| Application Number | Priority Date | Filing Date | Title |
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| CN 201110419568 CN102533591B (en) | 2011-12-15 | 2011-12-15 | High temperature resisting and high-glucose resisting lactic acid bacteria |
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| US10058577B2 (en) * | 2014-10-01 | 2018-08-28 | 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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| CN101880696B (en) * | 2010-07-14 | 2013-04-17 | 华中科技大学 | Method for producing L-lactic acid by fermentation and bacterial strain using same |
| CN102492735A (en) * | 2011-12-15 | 2012-06-13 | 天津工业大学 | Application of strain of high temperature and glucose resistant lactobacillus in lactate production |
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