CN102492735A - Application of strain of high temperature and glucose resistant lactobacillus in lactate production - Google Patents
Application of strain of high temperature and glucose resistant lactobacillus in lactate production Download PDFInfo
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- CN102492735A CN102492735A CN2011104199561A CN201110419956A CN102492735A CN 102492735 A CN102492735 A CN 102492735A CN 2011104199561 A CN2011104199561 A CN 2011104199561A CN 201110419956 A CN201110419956 A CN 201110419956A CN 102492735 A CN102492735 A CN 102492735A
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Abstract
The invention discloses application of a strain of high temperature and glucose resistant lactobacillus in lactate production, particularly relates to a high temperature and glucose resistant bacterial strain, and belongs to the field of lactobacillus. The lactobacillus is lactobacillus rhamnosus, the bacterial strain is filed in China General Microbiological Culture Collection Center with the filed number CGMCC No.4430, the bacterial strain CGMCC No.4430 can be resistant to the glucose of concentration reaching 270g/L which is increased by 95% as compared with that of original bacteria. After fermentation, the lactate content is 50g/L which is increased by 95% as compared with that of original bacteria. Various performance indexes of experiment tracks of a fermenting tank indicate that the bacteria strain is normal in metabolism, high in L-lactate production capability and low in heteroacid content and is a strain of excellent high temperature and glucose resistant lactobacillus rhamnosus.
Description
Technical field:
The invention belongs to newborn bacterium field, the lactobacillus strains of particularly high temperature resistant, anti-high sugar.
Background technology:
In recent years, because some special functions that have, 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 just material-POLYACTIC ACID of degradable macromolecule, 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 that each country extensively likes a kind of functional foodstuff that receives in the world, and they all are fermented bacterium with the milk-acid bacteria.Under senior staff officer and pyritous condition, 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 its molecule of base.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 made 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; Deposit number is 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 connect: 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.12134 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 original strain that the present invention adopted is the righttest, and to send out temperature pure be 37 ℃, in order to improve its resistant to elevated temperatures character, at first adopts and progressively improve 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 (55 ℃ of anti-changes) 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 arrive 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 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 the process that went down to posterity last time continuously, 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 20g, Tween 80 1.0g, potassium hydrogenphosphate 2.0g; MAGNESIUM SULPHATE HEPTAHYDRATE 99.5 0.2g, water manganous sulfate 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 107 mL bacteria suspensions with physiological water.
4) dilution is coated in the plane that contains substratum.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 107 mL bacteria suspensions with the slow liquid of pH7.0 phosphoric acid.
4) phosphoric acid 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 107 mL bacteria suspensions with the pH6.0 phosphoric acid buffer.
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) getting lactobacillus rhamnosus one ring that respectively tries on the inclined-plane on the super clean bench respectively, inserting 50mL culture medium A (no agar) glucose concn is housed is 250g/L) the 250mL triangular flask in, 200rpm, 30 cultivate about 15h, make 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 produce speed, glucose to last L-lactic acid transformation efficiency and heteroacid content.
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 was gone down to posterity on the inclined-plane continuously last time, and detected the fermentation situation after at every turn going down to posterity with the method for shaking the multiple sieve of bottle.Experiment is found, on the inclined-plane, goes down to posterity last time continuously, 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 rhamnosyl bacillus 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 production are 50g/L, compare with original bacterium and have improved 158%.
Give an example 2:
Other experimentation is with giving an example 1.
5L fermentor tank test: get rhamnosyl breast bacterium 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 60g/L) substratum, 200rpm cultivates about 12h for 30 ℃, makes thalline be in logarithmic growth mid-term.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 2 days.After the fermentation ends, no residual sugar, L-lactic acid content are 55g/L.
Claims (1)
1. the application of lactobacillus rhamnosus CGNCC No.4430 in lactic acid-producing of high temperature resistant, anti-high sugar.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102533591A (en) * | 2011-12-15 | 2012-07-04 | 天津工业大学 | High temperature resisting and high-glucose resisting lactic acid bacteria |
| 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 |
| CN109294940A (en) * | 2018-09-04 | 2019-02-01 | 湖南肯基因科技有限公司 | The purposes of corn lactobacillus mutagenic bacteria and high-yield lactic acid |
| CN111206006A (en) * | 2020-03-25 | 2020-05-29 | 福建傲农生物科技集团股份有限公司 | Screening and domesticating method of high-temperature-resistant enterococcus faecalis |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101880696A (en) * | 2010-07-14 | 2010-11-10 | 华中科技大学 | Method for producing L-lactic acid by fermentation and bacterial strain using same |
-
2011
- 2011-12-15 CN CN2011104199561A patent/CN102492735A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101880696A (en) * | 2010-07-14 | 2010-11-10 | 华中科技大学 | Method for producing L-lactic acid by fermentation and bacterial strain using same |
Non-Patent Citations (1)
| Title |
|---|
| YU L. ET AL.: "Genome shuffling enhanced L-lactic acid production by improving glucose tolerance of Lactobacillus rhamnosus", 《JOURNAL OF BIOTECHNOLOGY》, vol. 134, 11 January 2008 (2008-01-11) * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102533591A (en) * | 2011-12-15 | 2012-07-04 | 天津工业大学 | High temperature resisting and high-glucose resisting lactic acid bacteria |
| 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 |
| CN109294940A (en) * | 2018-09-04 | 2019-02-01 | 湖南肯基因科技有限公司 | The purposes of corn lactobacillus mutagenic bacteria and high-yield lactic acid |
| CN109294940B (en) * | 2018-09-04 | 2021-06-29 | 湖南肯基因科技有限公司 | Lactobacillus zeae mutant strain and application thereof in high yield of lactic acid |
| CN111206006A (en) * | 2020-03-25 | 2020-05-29 | 福建傲农生物科技集团股份有限公司 | Screening and domesticating method of high-temperature-resistant enterococcus faecalis |
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Application publication date: 20120613 |