CN1840653A - Mutant strain of lactobacillus casei, its preparation method and fermentation production of L-lactic acid - Google Patents
Mutant strain of lactobacillus casei, its preparation method and fermentation production of L-lactic acid Download PDFInfo
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Abstract
The invention relates to restructured bacterial strain (CGMCC No:1466) of Lactobacillus casei genome with fast acid production speed and stronger acid-resistance, the process for preparing the restructured bacterial strain and the production of L-acid from the bacterial strain through fermentation. The disclosed strain is named as Lactobacillus casei subsp. Rhamnosus.
Description
Technical field
The invention belongs to microbial technology field, be specifically related to preparation method and this bacterial strain that a kind of rate of producing acid reaches the strong lactobacterium casei genome of acid resistance reorganization bacterial strain, this reorganization bacterial strain soon and be used for fermentation production of L-lactic acid.
Background technology
Lactic acid is a kind of organic acid that is widely used in industries such as food, medicine, chemical industry, especially can be used for preparing biodegradable plastic-poly(lactic acid) with it as monomer, alleviates the energy and environmental problem, so the production of lactic acid obtains paying attention to day by day.Because the optical purity of lactic acid has material impact to the physical properties and the biodegradable rate of poly(lactic acid), the demand of high-optical-purity L-lactic acid increases day by day.Microbial fermentation can obtain the lactic acid of perfect optics purity, but, in Production by Microorganism Fermentation lactic acid process, because bacterial classification, substratum, all multifactor influences such as culture condition, fermentation and extracting method cause L-lactic acid that very big-difference is all arranged on output, yield and purity.Using production L-lactic acid bacterium commonly used at present has two big classes, and a class is a milk-acid bacteria, and another kind of is head mold (Rhizopus royzae).Root arrhizus fermentation belongs to aerobic heterofermentation, by glycolytic pathway, produces ethanol, fumaric acid etc., product complexity when fermentation produces L-lactic acid; The sugar transformation efficiency is lower.Lactobacillus-fermented is a homofermentation, can be 2mol lactic acid with the 1mol conversion of glucose, and theoretical yield is 100%; In addition, the milk-acid bacteria anaerobically fermenting can cut down the consumption of energy on a large scale, reduces the production cost of lactic acid.In recent years, the ability that has stronger acid resistance and can utilize genetically engineered to select to produce D-and L-lactic acid owing to Bacterium lacticum is widely used in lactic fermentation production, wherein lactobacterium casei rhamnosyl subspecies (L.caseisubsp.Rhamnosus) are only to produce the homotype lactic fermentation bacterial strain of L-lactic acid, also are the most frequently used bacterial strains of L-lactic acid-producing.But, using this bacterial strain to produce in the L-lactic acid process, there are two major issues, one is that rate of producing acid is low, the production cycle is long; Another is that the bacterial classification acid resistance is poor, can only be at nearly neutrallty condition bottom fermentation.Lactic fermentation is typical product inhibition type biotransformation, and the accumulation meeting cell growth inhibiting and the products production of free lactic acid need to add alkaline matters such as ammoniacal liquor and lime carbonate usually and neutralize, and fermentation system pH is maintained between the 6.0-6.5.It should be noted that the lactic acid original position that development in recent years gets up separates coupling connection fermentation technique, the lactic acid that promptly adopts method such as electrodialysis to remove in the reaction system is removed product inhibition.Screening can be in the microorganism that hangs down growth metabolism under the pH condition, especially be lower than below 3.8 (the lactic acid pKa=3.86) at pH, free lactic acid concentration accounts for the ratio of total lactic acid will be above 50%, can effectively reduce the pollution of assorted bacterium in the industrial production, simplify the product aftertreatment technology, reduce production costs, also help the development technology of lactic acid fermentation.Therefore, the acid resistance that improves Bacterium lacticum is an important development direction of improvement lactic acid-producing bacterial classification, receives the concern of Chinese scholars.People such as Patnaik adopt the genome shuffling technology, take turns reorganization through 5, and mutant strain can grow under pH 3.9 conditions, and at pH 4.0 condition bottom fermentations, lactic acid production has improved 3 times than the original strain bacterial strain.Human Kluyveromyces Lactis such as Porro express the lactate dehydrogenase gene engineering bacteria of ox and produce L-lactic acid at pH 4.5 condition bottom fermentations, but produce ethanol simultaneously, and transformation efficiency only is 59.5%, and fermenting process control is complicated, and by product is many.China University Of Science and Technology Of Tianjin has also carried out the lactic acid fermented research work of bacterium L-, obtains the bacterial strain that critical lactic acid concn is 24g/L through ion implantation mutagenesis, and the L-lactic acid production is 70g/L.
The sophisticated as a comparison gene manipulation techniques of genetically engineered is used comparatively extensive at present, be one and comprise the complex process with the reaction mechanism of a plurality of enzymes and gene-correlation but biological chemistry and proteomics and genetic analysis show that the acid of milk-acid bacteria is replied.Studies show that acid resistance of milk-acid bacteria and F
1F
0The macromole protection material a plurality of mechanism such as (comprising DNA and protein) that exist in the generation of alkaline matter and the cell in-ATPase, permeability of cell membrane, the cell are relevant.There are 63 species diversity marking proteins between two dimensional electrophoresis analysis revealed Lactobacillus sanfranciscensis acid resistance muton and the original strain.The optimization of metabolic engineering analysis revealed metabolic system needs the adjusting of a plurality of enzymes, except Gene Handling, may also need the participation of some non-coding regions.In addition, this research bacterial strain uses therefor is not also characterized by heredity.Therefore, directly improve its acid resistance and rate of producing acid is very difficult with molecular biology method.Utilize traditional mutafacient system,, need a large amount of time and manpower though can obtain the purpose bacterial strain.Genome reorganization (genome shuffling) technology is the evolution engineering new technology of being set up by people such as Patnaik (2002), promptly uses many female parents and passs nearly fusion (recursive fusion), and directed screening has the mutant that important phenotypic characteristic changes.Genome reorganization can be used for the improvement of the complicated phenotype of industrial microorganism as effective Research for Industrial Microbial Germ renovation technique.It should be noted that so far only the applying gene group has improved some proterties of bacterial strain, and it is big to merge back sudden change storage capacity, the screening operation amount is big, and screening time is long.Applying biological hi-tech means are carried out molecular breeding, obtain the two L-of sudden change lactic acid-producing bacterial classifications that acid resistance is strong and rate of producing acid is fast fast, and the operational path of setting up the L-lactic acid-producing has important practical significance.
Summary of the invention
One of purpose of the present invention is to be original strain with lactobacterium casei ATCC11443, by improvement genome shuffling technology and screening, obtains the L-lactic acid-producing bacterial strain that rate of producing acid is fast, optical purity is high and acid resistance is strong;
Two of purpose of the present invention provides the production method of the L-lactic acid-producing bacterial strain that a kind of above-mentioned rate of producing acid is fast, optical purity is high and acid resistance is strong;
Three of purpose of the present invention provides the method that strain fermentation that a kind of the present invention of utilization obtains is produced L-lactic acid.
Original strain--lactobacterium casei rhamnosyl subspecies ATCC11443 (hereinafter to be referred as Lc-WT) can be used for the production of L-lactic acid available from American type culture collection (ATCC).Optimum culturing temperature is 37 ℃, and pH generally is controlled between the 5.5-6.5 in the fermenting lactic acid process, needs a large amount of alkaline matter neutralizations in the fermenting process, has increased the operation of L-lactic acid downstream processing, has improved production cost.In addition, the lactic acid original position that new development is at present got up is separated coupling process can remove the inhibition of L-lactic acid in the fermented liquid, is an important research direction so improve the acid resistance of this bacterial strain.We find that in the previous experiments process throughput rate that improves this bacterial strain also has certain research space simultaneously, shorten the production cycle by improving throughput rate.In order to make this bacterial strain can better be used for the industry of L-lactic acid-producing, reduce the products production cost, it is significant to improve its acid resistance and throughput rate.All be difficult to obtain such two kinds of bacterial strains that complex character is undergone mutation simultaneously by genetically engineered and traditional mutafacient system.The present invention adopts the genome shuffling technology means of improvement, can address this problem.
Step of the present invention is:
The first step, the cultivation of original strain
Original strain-lactobacterium casei rhamnosyl subspecies ATCC11443 (to call Lc-WT in the following text) adopts the MRS solid medium to carry out separation and purification, colony inoculation bigger on the picking solid plate is in liquid MRS substratum, 37 ℃, 120 rev/mins, the concussion overnight incubation, 8000 rev/mins of centrifugal collection thalline, sterilization 0.01M TrisHCl (pH6.8) washed twice is resuspended among the sterilization 0.01M TrisHCl (pH 6.8);
Second step, the elementary mutagenesis of bacterial classification
After original strain was cultivated, (physical method was as ultraviolet (power is 15W), X ray and gamma ray to adopt physics and two kinds of different mutafacient system of chemistry respectively; Chemical process is as the sulfonic acid ethyl ester, ethyl sulfate, oxyethane and nitrosoguanidine) original strain is carried out mutagenic treatment (concrete mutafacient system is with reference to " microbiology study course " 244-250 page or leaf of Zhou Deqing chief editor), again by low pH flat board, lime carbonate flat board and shake flask test, respectively screening obtain through the rate of producing acid of physical method mutagenesis and acid resistance than original strain increase by 1% or more the plant mutant of manying bacterial strain-Lc-UV and through the rate of producing acid of chemical process mutagenesis and acid resistance than the many plant mutant bacterial strain-Lc-NTG of original strain increase more than 1%;
The 3rd step, genome reorganization (genome shuffling)
(1) protoplastis preparation and regeneration:
The mutant strain that yeast culture will be respectively obtains through ultraviolet ray and the elementary mutagenesis screening of NTG is in the MRS substratum after the incubated overnight, inoculation (inoculum size is 2～10%) contains 1.0～2.0% glycine liquid MRS substratum in 30ml respectively, 37 ℃ of shaking culture, 8000 rev/mins of centrifugal collection thalline.The thalline of collecting is washed 2 times with LPB, be resuspended among the LPB, add N,O-Diacetylmuramidase and mutanolysin, final concentration is respectively 1～20mg/mL and 5～50ug/mL, in 37 ℃ of enzymolysis 0.5～2.0h, microscopically is observed the broken wall situation, behind the 90% above thalline broken wall, centrifugal results protoplastis, LPB washing 2 times, be resuspended among the LPB standbyly, the protoplastis number average is 10 in the bacterium liquid
8The cfu/mL order of magnitude.
(2) to pass that nearly formula protoplastis merges be genome reorganization (genome shuffling) to many female parents
The ultraviolet mutagenesis that step (1) is obtained and the protoplastis bacterium liquid equal-volume of NTG mutagenic strain are (because the protoplastis number is a same order in the bacterium liquid that previous step obtains in rapid, so with protoplastis in the protoplastis bacterium liquid of each bacterial strain of volume is that the order of magnitude equates) be divided into two groups after the mixing, one group places power is the following 10～50min of processing of ultraviolet ray of 15W, after another group places 60 ℃ of water-baths to handle 30～150min complete inactivation, mix centrifugal, be resuspended among the 50 μ L LPB, the 40%PEG 6000 that adds 9 times of volumes, room temperature is placed 2～6min, add 5mL LPB dilution, centrifugal, washing, be resuspended in 100 μ L LPB, the multiple dilution is coated with the RM flat board, and 37 ℃, CO
2(5%) the incubator lucifuge was cultivated 5 days.The RM flat-plate bacterial colony is for merging the back bacterial strain.Bacterium colony on the RM flat board is washed with sterile saline, and dilution is coated with pH 4.0YE flat board, and 37 ℃, CO
2(5%) the incubator lucifuge is cultivated, and the pH 4.0 dull and stereotyped bacterium colonies of going up acquisition are washed with physiological saline, and suitably the dilution back is coated with lime carbonate flat board, 37 ℃, CO
2(5%) incubator is cultivated 48h, and the bacterium colony that transparent circle is bigger on the picking lime carbonate flat board carries out shake-flask culture 17h, measures and produces the acid amount, obtains producing the high mutant strain more than 1% of acid molar ratio original strain, and note is made Lc-F1;
Protoplastis preparation, deactivation, fusion and the screening (pH 3.8 flat boards, lime carbonate flat board and shake flask test) of next round are carried out in a plurality of Lc-F1 bacterial strains continuation steps (1), (2), measure the acid amount of producing, obtain producing the mutant strain of acid molar ratio Lc-F1 mutant bacteria plant height more than 2%, note is made Lc-F2;
Protoplastis preparation, deactivation, fusion and the screening (pH 3.6 flat boards, lime carbonate flat board and shake flask test) of next round are carried out in (1), (2) set by step will to screen the continuation of Lc-F2 bacterial strain once more, measure the acid amount of producing, obtain producing the mutant strain of acid molar ratio Lc-F2 mutant bacteria plant height more than 2%, note is made Lc-F3.
Each bacterial strain of Lc-F3 to above-mentioned acquisition carries out stability test, acid resistance and product acid amount property analysis, obtain the strongest and the fastest mutant strain (Lc-F34) of rate of producing acid of a strain acid resistance, adopt the high performance liquid chromatography result to show, not producing other organic acids in this bacterial strain fermentation liquor produces, lactic acid is main tunning still, and transformation efficiency is higher than 90%; L-lactic acid optical purity is higher than 95% in its fermented liquid of Boehringer Mannheim kit measurement.This bacterial strain has been preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on September 27th, 2005, the address is: No. 13, North No.1 Row, Zhongguancun, Haidian District, Beijing City (100080), deposit number is CGMCC No.1466, classification called after: lactobacterium casei rhamnosyl subspecies Lactobacillus casei subsp.rhamnosus.
The feature of CGMCC No.1466 bacterial strain provided by the invention is as follows:
1. morphological features: thalline is that crooked shape is shaft-like, Gram-positive, no gemma.
2. cultural characteristic: bacterium colony is an oyster white, obvious projection is arranged, neat in edge.Liquid culture can be carried out in common shaking table, the fermented liquid muddiness, and liquid level does not produce film.
3. physiological and biochemical property: homofermentation, produce L-lactic acid; Can utilize glucose, fructose, lactose, semi-lactosi, rhamnosyl, seminose, trehalose, N.F,USP MANNITOL, sorbyl alcohol, Sunmorl N 60S as sole carbon source; The catalase feminine gender; Gelatin does not liquefy; Milk does not peptonize; Do not reduce nitrate; Hydrolyzed starch not; Growth pH value is 3.2～9.5; Growth temperature is 5～50 ℃.
4. strain culturing condition: the dull and stereotyped cultivation needs carry out in 5% CO2gas incubator, and liquid culture can be carried out in common shaking table, 40 ℃ of the suitableeest culture temperature, optimal pH 5.5.
5. substratum:
(SHARPE) nutrient agar comprises peptone 10.0g to solid purifying substratum: MRS for MAN, ROGOSA, meat medicinal extract 10.0g, yeast extract 5.0g, glucose 20.0g, Sodium acetate trihydrate crystal 5 .0g, Tween 80 1.0mL, Triammonium citrate 2.0g, K
2HPO
42.0g, agar 15.0g, MgSO
47H
2O0.2g, MnSO
42H
2O 0.05g, distilled water 1000mL.Also can buy by ATCC.
Liquid proliferated culture medium: MRS liquid nutrient medium (not containing agar).Also can buy by ATCC.
Fermention medium: carbon source comprises one or more of glucose, sucrose, maltose, dextrose syrup (Changchun great achievement industry group) and lactose; Nutritive substance comprises that (corn steep liquor is brown, sticky shape liquid or dry powder, and corn steep liquor is handled by economic benefits and social benefits or triple-effect evaporator can make corn steep liquor for yeast extract, wheat root, wort, hair hydrolysis liquid, soybean protein hydrolysis liquid and corn steep liquor.Protein content is 20% in the Dried Corn Steep Liquor Powder, and amino acid is 12%; Contain protein 16%～30% in the liquid corn steep liquor, amino acid 8%～12%) one or more.
Show that according to the 8th edition authentication method that provides of " uncle outstanding systematic bacteriology handbook " (Bergy ' s Manual of Systematic Bacteriology) and above-mentioned test-results above-mentioned bacterial strains still belongs to lactobacterium casei rhamnosyl subspecies.
The present invention adopts parents' protoplastis of deactivation to merge the complementary method that obtains active fusant again, passing nearly formula protoplastis merges, improved the screening efficiency after the genome reorganization (genome shuffling) effectively, for the widespread usage of genome shuffling technology provides new way.
The present invention takes turns mutant strain to each and carries out two phenotype (acid resistance and rate of producing acid) screenings, obtain to carry out genome reorganization (genome shuffling) behind a plurality of parent's mutant strains, make the gain mutant characteristic altitude combination of bacterial strain, screen the L-lactic acid-producing bacterial strain that rate of producing acid is fast, acid resistance is strong, optical purity is high.
The present invention adopts low pH flat board, and (each takes turns the pH difference of the used low pH flat board in reorganization back, be followed successively by 4.2,4.0,3.8,3.6) and dull and stereotyped two kinds of simple prescreening methods of primary dcreening operation of lime carbonate and a method of shaking the multiple sieve of bottle, the strain excellent that seed selection acid resistance two phenotypes strong and that rate of producing acid is fast are suddenlyd change.Concrete grammar is as follows:
1. plate screening
Regeneration culture medium (RM) flat board: solid MRS substratum does not contain 2.5% gelatin in (not containing Tween80), the MgCl of 20mM
2, the sucrose of 0.5M, 115 ℃ of sterilizations sterilization in 20 minutes is cooled to 50 ℃, adds 0.5% calf serum, falls dull and stereotyped.With the protoplastis diluent coating regenerated plate of 40% polyoxyethylene glycol (PEG), 6000 processing after 6 minutes, CO
2(5%) 37 ℃ of cultivations of incubator, observations after 5 days.
Low pH value YE plate screening: yeast extract 15.0g, glucose 100.0g, agar 20.0g, distilled water 1000mL, 4M hydrochloric acid is regulated pH to 4.2, and 4.0,3.8 and 3.6,115 ℃ of sterilizations sterilization in 20 minutes is cooled to 50 ℃ and falls dull and stereotyped.Fusant on the regenerated plate is transferred to low pH value YE flat board, CO
2(5%) 37 ℃ of cultivations of incubator, observations after 5 days.
Lime carbonate plate screening: yeast extract 15g, glucose 100g, CaCO
3Be 2g, distilled water 1000mL sterilized 20 minutes for 115 ℃, was cooled to 50 ℃ and fell dull and stereotyped.To hang down that bacterium colony scrapes off on the pH flat board, suitably coat the lime carbonate flat board, CO after the dilution
2(5%) 37 ℃ of cultivations of incubator, the 24h observations.
2. shake flask test yeast extract 15.0g, glucose 100.0g, agar 3.0g, distilled water 1000mL mixes, and gets 100mL solution respectively and places the 250mL triangular flask, adds 9g lime carbonate, 115 ℃ of sterilizations 20 minutes.Experimental strain overnight culture inoculation (inoculum size is 10%) is in containing the 100mL YE substratum 250mL Erlenmeyer flask of (containing 9 gram lime carbonate), 37 ℃, 120 rev/mins of shaking culture.Between 15h～24h, sampling and measuring lactic acid content per hour.
Analytical procedure used in the present invention: comprise lactic acid, calcium lactate, glucose and biomass analysis.
(1) lactic acid: total lactic acid adopts day island proper Tianjin LC-9A high-efficient liquid phase chromatogram technique analysis in the fermented liquid, chromatographic condition: SCR-101H organic acid post, and the SPD-6AV UV-detector, wavelength 210nm, moving phase is 10mmol HClO
4L-and D-lactic acid adopt Boehringer Mannheim test kit to analyze.
(2) quantitative assay of calcium lactate: fermented liquid is through 8000rpm, and centrifugal 5min gets supernatant liquor 1mL in the distilled water of 100mL, adds the NaOH 10mL of 1M, with 2 of calconcarboxylic acids, uses 0.05M EDTANa
2Reagent titration, terminal point are that solution colour becomes pure blue.By EDTANa
2The content of volume calculation calcium lactate and lactic acid.The result is calculated as follows:
Calcium lactate content: W
CaL.2=218.2c*V (mg)
Lactic acid content: W
La=180c*V (g/L)
Wherein: the V-titration consumes EDTANa
2Amount (mL)
C-EDTANa
2Concentration (mol/L)
(3) glucose: adopt 3 ' 5-dinitrosalicylic acid (DNS) method to analyze.
(4) biomass: cell density is measured OD with ultraviolet spectrophotometer (cary50)
600To collect thalline after the fermented liquid, distilled water wash is resuspended in the distilled water, adjusts cell density OD
600Be about 10, get 50mL thalline solution and be dried to weight in 105 ℃, weigh and calculate.1OD
600Cell density is equivalent to the 0.35g/L dry cell weight.
The invention still further relates to a kind of method of the L-of production lactic acid: at first adopt the enlarged culturing of seed step by step method well known in the art to obtain the pure growth of Lc-F34 mutant strain, be inoculated in the sealed fermenting equipment that contains fermention medium by the inoculum size of 2%～20% fermention medium volumetric concentration pure growth again the Lc-F34 mutant strain, comprise the primary carbon source of 2%～30% volumetric concentration and the nitrogenous source of 1%～15% volumetric concentration in the fermention medium, all the other are water; Fermented liquid carries out anaerobically fermenting under 35 ℃～50 ℃, 200rpm agitation condition cultivates, and the pH value of control fermented liquid is 3.2～7.0 in the fermenting process, and 15h～120h ferments.
In concrete fermenting process, for obtaining ferment effect preferably, obtain higher productive rate, can carry out fed-batch fermentation, on above-mentioned zymotechnique basis, need add carbon source and nitrogenous source, make carbon source volumetric concentration in the fermented liquid maintain the fermented liquid cumulative volume 1%～20% between, the volumetric concentration of nitrogenous source maintain the fermented liquid cumulative volume 1%～10% between; Adding the carbon source time is 10h～20h; Adding the nitrogenous source time is 8h～20h.
Use the pH value of alkaline matter control fermented liquid in the fermenting process, employed alkaline matter comprises NaOH, ammoniacal liquor, CaCO
3In one or more.
Nutrition scopes such as carbon source that Lc-F34 bacterial strain of the present invention utilized and nitrogenous source are not limited to any specified carbohydrate and nitrogenous class material, can as long as can be utilized and transform the material that forms lactic acid by this bacterial strain.Being fit to carbon source of the present invention comprises: glucose, sucrose, maltose, dextrose syrup (Changchun great achievement industry group) and lactose, suitable nitrogenous source comprises: ammonium sulfate, yeast extract (Yeast extract), ammonium nitrate, corn steep liquor and other trophic factor-sal epsom, manganous sulfate, Trisodium Citrate, sodium acetate, rice bran.
In preferred embodiments, the present invention uses glucose, sucrose, and lactose is as carbon source; Ammonium sulfate, ammonium nitrate, extractum carnis, peptone, yeast extract and corn steep liquor are nitrogenous source; Further in the preferred embodiment, the present invention uses glucose as carbon source; Yeast extract and corn steep liquor are nitrogenous source; Carbon source adopts D-glucose in the optimum implementation of the present invention, and corn steep liquor is a nitrogenous source.
Further, the present invention adopts the response surface analysis method, in 5 liters of fermentor tanks, carry out bacterial strain Lc-F34 Optimizing Conditions of Fermentation of the present invention experiment, determine processing parameters such as its fermentation time, temperature, stirring velocity, and best feed supplement time in the fed-batch fermentation process.
Lc-F34 inoculation amount of the present invention is 2%～20% (volume ratio), and further in the preferred embodiment, it is 5%～15% that the present invention uses inoculum size, and inoculum size is 10% in the optimum implementation of the present invention.
The pH of fermenting process of the present invention is 3.2～7.0, and leavening temperature is 35 ℃～50 ℃; Further in the preferred embodiment, it is 4.5～6.0 that the present invention controls pH, and controlled temperature is 37 ℃～42 ℃; Control pH is 5.5 in the optimum implementation of the present invention, and controlled temperature is 40 ℃.
It is 2%～30% that the present invention adopts primary carbon source concentration, and further in the preferred embodiment, it is 8%～20% that the present invention uses primary carbon source concentration, and primary carbon source concentration is 10%～16% in the optimum implementation of the present invention.
It is 1%～15% that the present invention adopts initial nitrogen concentration, and further in the preferred embodiment, the present invention uses that initial nitrogen concentration-yeast extract is 1.0%～2.5%, corn steep liquor 4.5%～7.0%; Nitrogen concentration-yeast extract is 1.5～2.0% in the preferred forms, corn steep liquor is 5.0～6.5%.
Description of drawings
Fig. 1 (A): mutant strain storehouse and original strain compare on low pH 4.4 value YE flat boards;
Fig. 1 (B): mutant strain storehouse and original strain compare on low pH 3.6 value YE flat boards;
Fig. 2: reorganization bacterial strain and original strain bacterial strain are containing CaCO
3The YE shake flask fermentation is figure as a result;
Fig. 3 (A): mutant strain and the original strain comparison diagram of in pH 3.8 YE substratum, growing;
Fig. 3 (B): mutant strain and original strain produce sour comparison diagram in pH 3.8 YE substratum;
Fig. 4 (A): reorganization bacterial strain and the shake flask fermentation process of growth comparison diagram of original strain under pH 3.8 conditions;
Fig. 4 (B): reorganization bacterial strain and the shake flask fermentation acid process comparison diagram of original strain under pH 3.8 conditions;
Fig. 5: optimization of fermentation conditions response surface method stereoscopic analysis figure;
Fig. 6: 5L fermentor tank fed-batch fermentation is figure as a result;
Fig. 7: the fed-batch fermentation of 30L fermentor tank is figure as a result;
Reorganize bacterial strain and original strain fermentation comparison diagram as a result under Fig. 8: pH 4.5 conditions;
Reorganize bacterial strain and original strain fermentation comparison diagram as a result under Fig. 9: pH 5.0 conditions.
As shown in Figure 1,1 expression original strain Lc-WT wherein; 2 expression ultraviolet mutagenesis strain storehouse Lc-UV (unscreened hybrid bacterial strain behind the ultraviolet mutagenesis); 3 expression nitrosoguanidine mutagenesis strain storehouse Lc-NTG (unscreened hybrid bacterial strain after the NTG mutagenesis); 4 expression third round reorganization strain storehouse Lc-F3 (the unscreened hybrid bacterial strain in third round reorganization back).
As shown in Figure 2, reorganization bacterial strain and original strain are respectively at YE shake-flask culture 17h, the comparison of lactic acid production.WT represents original strain Lc-WT; UV represents ultraviolet mutagenesis bacterial strain Lc-UV1, Lc-UV2 successively; NTG represents NTG mutagenic strain Lc-NTG1, Lc-NTG2, Lc-NTG3 successively; F1 represents first round reorganization bacterial strain Lc-F11-16 successively; F2 represents that successively second takes turns reorganization bacterial strain Lc-F21-24; F3 represents third round reorganization bacterial strain Lc-F31-34 successively.
As shown in Figure 3, mutant strain and original strain bacterial strain were cultivated 48 hours respectively at pH 3.8 YE liquid shaking bottles, in the different time sampling, measured OD in the fermenting process
600And lactic acid production.WT represents original strain Lc-WT; UV represents ultraviolet mutagenesis bacterial strain Lc-UV1,2 successively; NTG represents NTG mutagenic strain Lc-NTG1-3 successively; F1 represents first round reorganization bacterial strain Lc-F11-16 successively; F2 represents that successively second takes turns reorganization bacterial strain Lc-F21-24; F3 represents third round reorganization bacterial strain Lc-F31-34 successively.
As shown in Figure 4, third round reorganization bacterial strain and original strain be respectively in 48 hours processes of shake-flask culture among the pH 3.8 liquid YE, cell density (OD
600) with the comparison of lactic acid production.Curve 0 expression original strain Lc-WT; Curve 1 expression reorganization bacterial strain Lc-F31; Curve 2 expression reorganization bacterial strain Lc-F32; Curve 3 expression reorganization bacterial strain Lc-F33; Curve 4 expression reorganization bacterial strain Lc-F34.
As shown in Figure 5, using bacterial strain Lc-F34 of the present invention, to prepare in the L-milk-acid bacteria technology suitable leavening temperature be 37～42 ℃, and suitable pH is 5.0～6.0; The suitableeest leavening temperature is 40 ℃, and optimal pH is 5.5.
As shown in Figure 6, for preparing, application bacterial strain Lc-F34 of the present invention adopts 5L fermentor tank fed-batch fermentation result in the L-milk-acid bacteria technology.Lc-F34 is with corn steep liquor and dextrose culture-medium (first sugared concentration is 10%), control rotating speed 200rpm, and temperature is 40 ℃, with in the lime carbonate and the lactic acid that produces in the fermenting process, fermentation 12h adds glucose 200.0g, corn steep liquor 60.0g, fermentation 26h.Curve 1: residual sugar; Curve 2: dry cell weight; Curve 3: lactic acid production.
Shown in Figure 7, the fed-batch fermentation result of 30L fermentor tank.Lc-F34 is with corn steep liquor and dextrose culture-medium (first sugared concentration is 10%), control rotating speed 200rpm, and temperature is 40 ℃, with in the lime carbonate and the lactic acid that produces in the fermenting process, fermentation 24h adds glucose 1600.0g, fermentation 40h.Curve 1: residual sugar; Curve 2: lactic acid production.
Shown in Figure 8, reorganization bacterial strain and original strain fermentation result relatively under pH 4.5 conditions.Lc-WT and Lc-F34 cultivate respectively at the 5L ferment tank, and fermention medium is a liquid YE substratum, and working volume is 3L, with the NH of 7.0M
4OH is controlled to be 4.5 respectively with pH, fermentation 84h, the variation of cell density and lactic acid production in the monitoring fermenting process.Curve 1:Lc-F34 bacterial strain residual sugar; Curve 2:Lc-F34 bacterial strain dry cell weight; Curve 3:Lc-F34 bacterial strain lactic acid production.Curve 4:Lc-WT bacterial strain residual sugar; Curve 5:Lc-WT bacterial strain dry cell weight; Curve 6:Lc-WT bacterial strain lactic acid production.
Shown in Figure 9, reorganization bacterial strain and original strain fermentation result relatively under pH 5.0 conditions.Lc-WT and Lc-F34 cultivate respectively at the 5L ferment tank, and fermention medium is a liquid YE substratum, and working volume is 3L, pH are controlled to be 5.0 respectively, fermentation 84h, the variation of cell density and lactic acid production in the monitoring fermenting process with the NH4OH of 7.0M.Curve 1:Lc-F34 bacterial strain residual sugar; Curve 2:Lc-F34 bacterial strain dry cell weight; Curve 3:Lc-F34 bacterial strain lactic acid production.Curve 4:Lc-WT bacterial strain residual sugar; Curve 5:Lc-WT bacterial strain dry cell weight; Curve 6:Lc-WT bacterial strain lactic acid production.
Embodiment
The seed selection of embodiment 1:L-lactobacillus inoculation
(1) the elementary mutagenesis of bacterial classification
The present invention adopts ultraviolet ray (UV) and nitrosoguanidine (NTG) to carry out following mutagenesis respectively to original strain:
Ultraviolet ray (UV) mutagenesis: original strain adopts the MRS solid medium to carry out separation and purification, colony inoculation bigger on the picking solid plate is in liquid MRS substratum, 37 ℃, 120 rev/mins, the concussion overnight incubation, 8000 rev/mins of centrifugal collection thalline, sterilization 0.01M TrisHCl (pH 6.8) washed twice, be resuspended in sterilization 0.01MTrisHCl (pH 6.8), adjusting cell concentration is 10
8Cfu/mL.10mL thalline solution is placed the culture dish (diameter 90mm) of the band magnetic bar of sterilization (160 ℃ of dry sterilization box sterilization 2h), culture dish is placed on the magnetic stirring apparatus Stage microscope, turn on agitator, (power is 15W at the ultraviolet lamp of preheating 20min, apart from 20cm) stir on following irradiation limit, and timing.Respectively at 0.5min, 1.0min, 1.5min, 3.0min, 5.0min respectively get 2mL bacterium liquid and mix, and after suitably diluting, coat pH 4.2 YE flat boards, CO
2(5%) 37 ℃ of cultivations of incubator, observations after 5 days.Bacterium colony on the pH 4.2 YE flat boards is washed with sterile saline, and suitably the dilution back is coated with lime carbonate flat board, CO
2(5%) incubator is cultivated 48h for 37 ℃, and the bigger bacterium colony of transparent circle carries out shake flask fermentation on the picking lime carbonate flat board, and the 17h sampling and measuring is produced the acid amount.Obtain two strains and produce higher bacterial strain-Lc-UV1 and the Lc-UV2 of acid amount.
Nitrosoguanidine (NTG) mutagenesis: original strain is cultivated by method described in the ultraviolet mutagenesis, and is centrifugal, and thalline is collected in washing, is resuspended among the 0.01M TrisHCl (pH 6.0), and adjusting cell concentration is 10
8Cfu/mL.Get 10mL bacterium liquid and be divided into 5 parts, adding NTG respectively is 50 μ g/mL to final concentration, 100 μ g/mL, 200 μ g/mL, 500 μ g/mL, 1000 μ g/mL place 30 ℃ of following incubated overnight, and are centrifugal, washing, carry out the multiple dilution behind the mixing, screen, obtain three strain NTG mutagenic strain-Lc-NTG1, Lc-NTG2 and Lc-NTG3 by screening method behind the ultraviolet mutagenesis.
Above mutagenesis gained 5 plant mutant bacterial strains are the initial bacterial strain that is used for the genome reorganization.
(2) genome reorganization (genome shuffling)
A. protoplastis preparation and regeneration with 5 bacterial strains of the ultraviolet ray of preliminary screening and NTG mutagenesis in the MRS substratum after the incubated overnight, inoculation (inoculum size is 10%) contains 1.0% glycine MRS liquid nutrient medium in 30mL respectively, 37 ℃ of shaking culture 12h, centrifugal collection thalline.The thalline of collecting (is contained 20mM MgCl2 and 0.5M sucrose with LPB among the 0.01M TrisHCl, pH 6.8) wash 2 times, be resuspended among the LPB, add N,O-Diacetylmuramidase (Lysozyme Beijing ancient cooking vessel state biotechnology limited liability company provides) and mutanolysin (Mutannolysin simultaneously, Sigma company provides), final concentration is respectively 10mg/mL and 25ug/mL, and in 37 ℃ of enzymolysis 1.0～1.5h, microscopically is observed the broken wall situation.Behind the 90% above thalline broken wall, centrifugal results protoplastis, LPB washing 2 times is resuspended among the LPB standby.
B. many female parents are passed nearly formula protoplastis and are merged that to be genome reorganization (genome shuffling) mix back (the protoplastis quantity that guarantees each bacterial strain equates) with the protoplastis solution equal-volume of the 5 strain ultraviolet mutagenesis bacterial strains that prepare in the steps A and NTG mutagenic strain is divided into two groups, one group places ultraviolet ray down (power is 15W, apart from ultraviolet lamp 20cm) processing 15min, after another group places 60 ℃ of water-baths to handle the 50min complete inactivation, protoplastis after two groups of deactivations mixes centrifugal, be resuspended among the 50 μ L LPB, the 40%PEG 6000 that adds 9 times of volumes, room temperature is placed 2min, add 5mL LPB dilution, centrifugal, washing, be resuspended in 100 μ L LPB, the multiple dilution is coated with the RM flat board, and 37 ℃, CO
2(5%) the incubator lucifuge was cultivated 5 days.Bacterium colony on the RM flat board is washed with sterile saline, and dilution is coated with pH 4.0 YE flat boards, and 37 ℃, CO
2(5%) the incubator lucifuge is cultivated, and through pH 4.0 flat boards, lime carbonate flat board and shake flask test screening, obtains 6 strains and produces the mutant strain that the acid amount is high, acid resistance is strong, i.e. first round recombinant bacterial strain Lc-F11, Lc-F12, Lc-F13, Lc-F14, Lc-F15, Lc-F16.6 F1 bacterial strains of gained are cultivated by same strategy, the protoplastis preparation is merged, and the regeneration back is in pH 3.8 YE flat boards, lime carbonate flat board and shake flask fermentation experiment screening, obtain second and take turns recombinant bacterial strain (F2) 4 strains, note is made Lc-F21, Lc-F22, Lc-F23, Lc-F24; Once more 4 strain F2 bacterial strains are merged, screen (pH 3.6 flat boards, the dull and stereotyped and shake flask fermentation experiment screening of lime carbonate) and obtain F3 bacterial strain, Lc-F31, Lc-F32, Lc-F33, Lc-F34.Taking turns the protoplast regeneration bacterial strain with each that handle without PEG compares.
(3) genetic stability of mutant strain experiment
Third round is reorganized bacterial strain carries out the MRS liquid nutrient medium respectively and go down to posterity and cultivate 20 times, coating pH 3.6 YE flat boards, by calculate viable count relatively its acid resistance on flat board change; Simultaneously, carry out shake flask fermentation experiment (37 ℃ of concussions are cultivated, and the 17h sampling and measuring is produced the acid amount) respectively, relatively its rate of producing acid (table 1).The result as seen, mutant strain is through 20 cultivations of going down to posterity, noticeable change does not take place in acid resistance and shake flask fermentation volume productive rate on solid plate, shows that mutant strain has good genetic stability.
Table 1. mutant strain stability experiment result
| Bacterial strain | Viable count (cfu/mL) | Rate of producing acid (g/L/h) | ||
| The 1st generation | The 20th generation | The 1st generation | The 20th generation | |
| Lc-F31 | 3.26*10 8 | 3.57*10 8 | 5.68 | 5.71 |
| Lc-F32 | 2.97*10 8 | 2.61*10 8 | 5.79 | 5.66 |
| Lc-F33 | 3.15*10 8 | 3.84*10 8 | 5.62 | 5.46 |
| Lc-F34 | 3.37*10 8 | 3.52*10 8 | 5.81 | 5.79 |
Embodiment 2: the comparison of mutant strain and original strain
(1) mutant strain and the original strain comparison on low pH YE flat board
With ultraviolet ray (Lc-UV), NTG (Lc-NTG) and third round reorganization (Lc-F3) mutant strain storehouse and original strain Lc-WT are respectively at cultivating (Fig. 1) on pH 4.4, the 3.6 YE flat boards.Lc-UV, Lc-NTG and Lc-F3 strain storehouse and Lc-WT all can growth and breedings on pH 4.4 YE flat boards, and have only Lc-F3 to grow on pH 3.6 YE flat boards, and other bacterial strains (storehouse) can not be grown.
(2) comparison of mutant strain and original strain rate of producing acid
With the mutant strain of each step preparation and original strain (Lc-WT) respectively at containing 9%CaCO
3YE shakes in the bottle and fermented 17 hours, adopts the content of EDTA titration measuring Lactic Acid from Fermentation Broth.Its lactic acid-producing situation of comparison.Result (Fig. 2) shows, traditional mutafacient system (ultraviolet ray and NTG mutagenesis) obtains mutant strain (Lc-UV1,2 and Lc-NTG1-3) shake flask fermentation and cultivates 17h, and its lactic acid-producing amount is distinguished not remarkable than original strain; And reorganization bacterial strain (Lc-F11-16, Lc-F21-24 and Lc-F31-34) lactic acid production all is significantly improved than original strain, and third round reorganization bacterial strain improves more remarkable, and fermentation 17h produces the acid amount and improved 27% than wild strain.The volume productive rate of original strain is 4.55g/L/h, and the volume productive rate of third round fusant bacterial strain is between 5.72-5.82g/L/h.Get fermented liquid and carry out centrifugally, filter dilution.Adopt high performance liquid chromatography and Boehringer Mannheim test kit to analyze L-lactic acid purity.High performance liquid chromatography is the result show, do not produce other organic acids in the mutant bacteria, and lactic acid still is main tunning; L-lactic acid optical purity reaches 98.2% in the Boehringer Mannheim kit measurement reorganization bacterial strain fermentation liquor.The pathways metabolism that the reorganization bacterial strain is described does not change.
(3) mutant strain and original strain shake flask fermentation feature are relatively
Mutant strain and original strain shake flask fermentation result relatively with mutant strain and original strain respectively at cultivating 48 hours in the pH 3.8YE liquid shaking bottle, relatively its growth and lactic acid-producing situation (Fig. 3).The result shows that traditional mutafacient system obtains mutant strain (Lc-UV1-2 and Lc-NTG1-3) and cultivates 48h at pH 3.8 condition bottom fermentations, and its thalli growth and lactic acid-producing amount are not remarkable than the original strain difference; And reorganization bacterial strain (Lc-F11-16, Lc-F21-24 and Lc-F31-34) is that thalli growth amount or lactic acid production all are significantly improved than original strain, and third round reorganization bacterial strain improves more remarkable, fermentation 48h, original strain (Lc-WT) cell density (OD
600) be 1.69, lactic acid production only is 1.59g/L, and the cell density (OD of reorganization bacterial strain
600) reach about 3.4, all between 4.6-5.0g/L, best reorganization bacterial strain produces the acid amount and reaches 5.00g/L lactic acid production, is 3.14 times of original strain.It is shown in Figure 3,
(4) third round recombinant bacterial strain and original strain shake flask fermentation advance ratio are
Third round recombinant bacterial strain and original strain are inoculated in pH 3.8 YE liquid nutrient mediums respectively, 37 ℃ of fermentation 96h, cell density and lactic acid production changing conditions (Fig. 4) in the monitoring fermenting process.As seen from Figure 4, before the fermentation 24h, the growth of third round recombinant bacterial strain and Lc-WT (shown in figure A) has similarity with product acid (shown in figure B), and cell density and product acid amount there are differences again between each bacterial strain, but the cell density of third round recombinant bacterial strain and product acid amount are all apparently higher than original strain.Behind the fermentation 24h, original strain (Lc-WT) cell density slightly increases, and lactic acid production does not but have to change, and the cell density of reorganization back bacterial strain all presents tangible ascendant trend with product acid amount.Fermentation 96h, and the cell density (OD of reorganization bacterial strain
600) reach about 5.0, all between 4.7-5.67g/L, best reorganization bacterial strain produces the acid amount and reaches 5.67g/L lactic acid production, is 3.5 times of original strain.Illustrate that the reorganization bacterial strain still can carry out growth metabolism under acidic conditions, prove that fully the acid resistance of reorganization bacterial strain is improved significantly.Because Bacterium lacticum produces acid and mainly concentrates on logarithmic phase, improve bacterial classification and can effectively improve the output of lactic acid in the fermenting process in the product acid amount of stationary phase.96 hours pH of reorganization bacterial strain Lc-F31-34 fermentation are minimum can to reach 3.3, and original strain only reaches about 3.5.
Embodiment 3: Optimum of culture medium
In the L-lactic acid production process, raw-material price is an important factor in order of product cost.Milk-acid bacteria is harsh to nutritional requirement, need be nitrogenous source with the yeast extract usually, according to Tejayadi and Cheryan (1995) report, is that nitrogenous source is produced in the cost of L-lactic acid with yeast extract, and yeast extract accounts for 38% of total cost.The cheap fermention medium of screening is very important for the production cost that reduces L-lactic acid.Therefore, we adopt Placket-Burman design (a kind of statistics optimization experiment design method that this area is commonly used) method that nutritive substances such as glucose, corn steep liquor, rice bran are optimized, thereby obtain to be suitable for the fermention medium of scale operation.
(1) two hydraulic test is adopted Placket-Burman design (table 2) to genome reorganization bacterial strain Lc-F34
Table .2 Placket-Burman experimental design
| Sequence number | X 1 | X 2 | X 3 | X 4 | X 5 | X 6 | X 7 | X 8 | X 9 | X 10 | X 11 | X 12 | X 13 | X 14 | X 15 | Y (g/L) |
| 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 | +1 -1 -1 -1 -1 +1 +1 +1 -1 +1 +1 -1 -1 +1 +1 -1 | +1 +1 -1 -1 -1 -1 +1 +1 +1 -1 +1 +1 -1 -1 +1 -1 | +1 +1 +1 -1 -1 -1 -1 +1 +1 +1 -1 +1 +1 -1 -1 -1 | -1 +1 +1 +1 -1 -1 -1 -1 +1 +1 +1 -1 +1 +1 -1 -1 | -1 -1 +1 +1 +1 -1 -1 -1 -1 +1 +1 +1 -1 +1 +1 -1 | +1 -1 -1 +1 +1 +1 -1 -1 -1 -1 +1 +1 +1 -1 +1 -1 | +1 +1 -1 -1 +1 +1 +1 -1 -1 -1 -1 +1 +1 +1 -1 -1 | -1 +1 +1 -1 -1 +1 +1 +1 -1 -1 -1 -1 +1 +1 +1 -1 | +1 -1 +1 +1 -1 -1 +1 +1 +1 -1 -1 -1 -1 +1 +1 -1 | +1 +1 -1 +1 +1 -1 -1 +1 +1 +1 -1 -1 -1 -1 +1 -1 | +1 +1 +1 -1 +1 +1 -1 -1 +1 +1 +1 -1 -1 -1 -1 -1 | -1 +1 +1 +1 -1 +1 +1 -1 -1 +1 +1 +1 -1 -1 -1 -1 | -1 -1 +1 +1 +1 -1 +1 +1 -1 -1 +1 +1 +1 -1 -1 -1 | -1 -1 -1 +1 +1 +1 -1 +1 +1 -1 -1 +1 +1 +1 -1 -1 | -1 -1 -1 -1 +1 +1 +1 -1 +1 +1 -1 -1 +1 +1 +1 -1 | 91.8 55.8 64.8 50.4 52.2 73.8 66.6 90.0 50.4 100.8 91.8 63.0 54.0 86.4 79.2 37.8 |
Nutritive substances such as glucose, ammonium sulfate, yeast extract, ammonium nitrate, corn steep liquor, sal epsom, manganous sulfate, Trisodium Citrate, sodium acetate, rice bran are optimized, investigate the main effect and the interactive one-level effect (table 3) of each factor.Utilize SAS software (Statistics Analysis System, famous computer statistics software can be by online download) to analyze, the result shows that glucose, yeast extract and corn steep liquor produce acid to reorganization bacterial strain Lc-F34 remarkably influenced is arranged.
Table 3.Placket-Burman design screening and culturing based component result
| Medium component | Estimate | Pr＞|t| |
| Glucosamine sulphate ammonium yeast extract ammonium nitrate corn steep liquor magnesium sulfate manganese sulfate natrium citricum sodium acetate rice bran wheat bran syrup | 1.287 0.139 0.326 -0.081 0.262 0.021 -0.294 -0.103 -0.089 0.150 -0.042 -0.170 | 0.0006 0.1966 0.0314 0.4227 0.0566 0.8143 0.0382 0.3191 0.3795 0.1766 0.6526 0.1286 |
(2) corn steep liquor concentration is to producing the influence of L-lactic acid to the reorganization strain fermentation
With 2.5%, 3.5%, 4.0%, 5% and 5.5% corn steep liquor is a nitrogenous source respectively, and D-glucose is carbon source, and its concentration 100g/L carries out the shake flask fermentation test, studies the influence (table 4) that different corn steep liquor concentration are produced lactic acid to reorganization bacterial strain Lc-F34 batch fermentation.The result shows that corn steep liquor concentration is between 2.5%-5.0%, and the L-lactic acid concn increases with glucose concn.The fermentation of 5.0% and 5.5% corn steep liquor is basically identical as a result.Show that thus 5.0% corn steep liquor can provide enough nitrogenous sources, corn steep liquor is 5.0% as the nitrogenous source optimum concn.
Table 4. original corn slurry concentration is to the influence of reorganization bacterial strain Lc-F34 fermenting lactic acid
| The experiment sequence number | Substratum (g/L) | 24h lactic acid production (g/L) | |
| D-glucose | Corn steep liquor (liquid) | ||
| 1 2 3 4 5 6 | 100 100 100 100 100 100 | 2.5 3.5 4.0 4.5 5.0 5.5 | 41.34 62.5 78.68 89.36 98.43 97.38 |
(3) initial glucose sugar concentration is produced the influence of L-lactic acid to the reorganization strain fermentation
Be nitrogenous source with 5% corn steep liquor respectively, the D-glucose concn is 30g/L, 50g/L, 70g/L, 90g/L, 110g/L, 140g/L, 170g/L, 200g/L carry out the shake flask fermentation test, study the influence (table 5) that different initial glucose concentrations are produced lactic acid to reorganization bacterial strain Lc-F34 batch fermentation.The result shows that the L-lactic acid concn increases with glucose concn.Transformation efficiency is on a declining curve with the increase of glucose concn, the volume productive rate is subjected to first sugared concentration affects also very big, in the time of between first sugared concentration 30～110g/L, the volume productive rate increases gradually, when first sugared concentration is 110g/L, the volume productive rate reaches maximum (5.84g/L/h), and glucose concn continues to increase, and the volume productive rate reduces on the contrary.This is that osmotic pressure increases, and has influenced the normal growth and the metabolism of thalline because under the high glucose concentration.In order to obtain the production rate and the inversion rate of glucose of higher L-lactic acid, first sugared concentration can be controlled at 90～110g/L and carry out fed-batch fermentation.The feed supplement time should be controlled by glucose concn in the monitoring fermented liquid and control, and makes the total sugar concentration in the fermented liquid be no more than 110g/L for beneficial.
Table 5. initial glucose concentration is to the influence of reorganization bacterial strain Lc-F34 fermenting lactic acid
| Initial glucose (a/L) | Fermentation time (h) | Transformation efficiency (%) | Volume productive rate (a/L/h) | Lactic acid concn (a/L) |
| 30 50 70 90 110 140 170 200 | 7.0 10.0 13.5 16.0 18.5 25.0 44.0 61.5 | 99 98.8 97.9 98.1 98.0 97.4 90.2 87.7 | 4.24 4.90 5.07 5.56 5.84 5.4 3.11 2.27 | 29.7 49.0 68.5 89.0 108.0 136.4 142.6 156.1 |
(4) corn steep liquor and yeast extract fermentation production of L-lactic acid result are relatively
Adopt the 5L ferment tank to cultivate, the fermention medium nutritive substance is respectively yeast extract and corn steep liquor, and the D-glucose concn is 10%, and working volume is 3L, and inoculation Lc-F34 (inoculum size 10%) is with the NH of 7.0M
4OH is controlled to be 5.5 (tables 6) with pH.
Table 6. is the result that nitrogenous source Lc-F34 strain fermentation is produced L-lactic acid with yeast extract and corn steep liquor respectively
| Substratum | Yeast extract (1.5%) | Corn steep liquor (5%) |
| PH fermentation time (h) cell concn (OD 600) L-lactic acid (g/L) transformation efficiency (%) maximum volume productive rate (g/L/h) | 5.5 24 20.7 92.34 95.31 5.88 | 5.5 24 21.2 92.15 94.56 5.82 |
Replacing yeast extract with corn steep liquor is nitrogen source fermentation 17h, cell density and lactic acid yield, and transformation efficiency and maximum volume productive rate and yeast extract are basically identical as a result of identical time of nitrogen source fermentation.Therefore, corn steep liquor can be used as nitrogenous source and is used for lactic fermentation production.
Embodiment 4:Lc-F34 bacterial strain fed-batch fermentation is produced lactic acid
Adopt reorganization bacterial strain Lc-F34 fed-batch fermentation to produce lactic acid technology in order further to study, bottle (yeast extract 1.5% is shaken in employing, D-glucose starting point concentration is 100g/L, the 250mL triangular flask, liquid amount 100mL) fermentation, and add D-glucose 100g/L in different time in batches, fermentation is (residual D-glucose is that 1g/L is following) to terminal, sampling and measuring lactic acid content (seeing Table 7).By table 7 as seen, Lactic Acid from Fermentation Broth concentration and total fermentation time change with the feed supplement asynchronism(-nization), and the feed supplement time is controlled between 12～15h, and lactic acid production is the highest, and fermentation time is the shortest.
Table 7: the different feed supplement times are produced the influence of lactic acid to the Lc-F34 strain fermentation
| Add D-glucose (g/L) | The feed supplement time (h) | Residual D-glucose (g/L) before the feed supplement | Total fermentation time (h) | Transformation efficiency (%) | Volume productive rate (g/L/h) | Lactic acid concn (g/L) |
| 100 100 100 100 100 100 100 | 10 11 12 13 14 15 16 | 55.1 49.7 43.4 36.3 28.4 21.1 12.6 | 50 48 45 45 45 46 47 | 85.5 88.3 94.8 94.5 95.5 92.8 91.6 | 3.40 3.66 4.19 4.18 4.22 4.01 3.87 | 159.1 164.2 176.4 175.7 177.6 172.6 170.4 |
Embodiment 5: fermentation condition optimization
Adopting the 5L ferment tank to cultivate, is fermention medium with liquid YE substratum, and working volume is 3L, with the NH of 7.0M
4OH controls pH, and stirring velocity is 200rpm, and temperature and pH are by designing the 17h that ferments in the table 8.
Table 8. reorganization bacterial strain Lc-F34 leavening temperature and the design of pH optimization Test
| Variable | -a | -1 | 0 | +1 | +a |
| Temperature (℃) pH | 32.9 4.25 | 35 4.5 | 40 5.5 | 45 6.5 | 47.1 6.75 |
Application responds surface analysis (Response Surface Methodology, a kind of statistics optimisation technique) is optimized-leavening temperature and pH value the optimal conditions of fermentation of reorganizing the back bacterial strain, operation SAS software analysis experimental result (Fig. 5).Resulting match entire variable quadratic regression equation is:
Y＝68.7530+7.2897pH+10.9896T-8.3966pH*pH+0.125T*pH-12.1477T*T
By the result as can be known, coefficient of determination R
2=0.8935, illustrate that the fitting degree of regression equation is better.Temperature and pH are the important factors (p=0.0326 and 0.0418) that influence reorganization bacterial strain Lc-F34 produces L-lactic acid.As shown in Figure 5, suitable leavening temperature is 37～42 ℃, and pH is 4.5～6.0, and the most suitable pH is 5.5, and temperature is 40 ℃.
Embodiment 6: adopt low capacity fermentor tank fed-batch fermentation to produce lactic acid
Adopt the 5L fermentor tank, initial medium is yeast extract (1.5%) and D-dextrose culture-medium (first sugared concentration is 10%), the initialization volume is 2.5L, inoculation 10%Lc-F34 nutrient solution, control rotating speed 200rpm, temperature is 40 ℃, with in the lime carbonate (adding 9% sterilization lime carbonate after sterilization in the substratum) and the lactic acid (pH is controlled at 4.5-6.0) that produces in the fermenting process, fermentation 10h adds yeast extract 30.0g; Fermentation 12h, residual sugar reduces to 4%, adds D-glucose 200.0g, ferments to 26h, and the fermented liquid cumulative volume is 3.36L, and residual sugar is 1.02%, and the L-concentration of lactic acid is 118.38g/L, and transformation efficiency is 95.7%, and the average-volume productive rate is 4.55g/L/h (Fig. 6).Conventional lactic acid-producing must be added alkaline matters such as lime carbonate or ammoniacal liquor and be neutralized, otherwise too much lactic acid can suppress thalli growth.
Embodiment 7: adopt large vol fermentor tank fed-batch fermentation to produce lactic acid
Adopt the 30L fermentor tank, with corn steep liquor (5%) and D-glucose is fermented substrate, first sugared concentration is 16%, the initialization volume is 20L, inoculation 10%Lc-F34 nutrient solution, control rotating speed 200rpm, temperature is 40 ℃, with in the lime carbonate (adding 9% sterilization lime carbonate after sterilization in the substratum) and the lactic acid (pH is controlled at 4.5-6.0) that produces in the fermenting process.Fermentation 15h adds corn steep liquor 800g; Behind the fermentation 24h, add D-glucose 1600.0g, continue to ferment to 40h, the fermented liquid cumulative volume is 26.4L, and residual sugar is 0.10%, and the L-concentration of lactic acid is 154.6g/L, and transformation efficiency is 91%, and the average-volume productive rate is 3.85g/L/h (Fig. 7).
Embodiment 8: low pH fermentation production of L-lactic acid
The minimum growth pH of Lc-WT bacterial strain is 4.4, adopts liquid YE culture medium culturing Lc-WT and Lc-F34 near the minimum growth pH (4.5) of original strain, relatively the growth and the production feature of two bacterial strains.The acid resistance superior strain Lc-F34 and the Lc-WT bacterial strain of third round reorganization back screening are cultivated respectively at the 5L ferment tank, and fermention medium is a liquid YE substratum, and working volume is 3L, with the NH of 7.0M
4OH is controlled to be 4.5,5.0 respectively with pH, the variation of cell density and lactic acid production in the monitoring fermenting process.Under the condition of pH 4.5 (Fig. 8), ferment to 84h the dry cell weight 4.54g/L that the Lc-F34 bacterial strain obtains, residual sugar is 3.82g/L in the fermented liquid, and lactic acid concn 83.8g/L, transformation efficiency are 86%, the average volume productive rate is 0.998g/L/h, maximum volume productive rate 3.59g/L/h; And the dry cell weight 3.59g/L that the Lc-WT bacterial strain obtains, Lactic Acid from Fermentation Broth concentration 52.17g/L, residual sugar are 43.1g/L, and transformation efficiency is 56.2%, and the average volume productive rate is 0.623g/L/h, maximum volume productive rate 3.02g/L/h.Lactic acid concn has improved 60.6% than original strain in the Lc-F34 bacterial strain fermentation liquor, and the average volume productive rate has improved 60.2%.
Under the condition of pH 5.0 (Fig. 9), ferment to 60h the dry cell weight 6.34g/L that the Lc-F34 bacterial strain obtains, residual sugar is 0.93g/L in the fermented liquid, and lactic acid concn 88.06g/L, transformation efficiency are 89.6%, the average volume productive rate is 1.47g/L/h, maximum volume productive rate 5.17g/L/h; And the dry cell weight 4.36g/L that the Lc-WT bacterial strain obtains, Lactic Acid from Fermentation Broth concentration 61.75g/L, residual sugar are 43.1g/L, and transformation efficiency is 74.9%, and the average volume productive rate is 1.03g/L/h, maximum volume productive rate 4.11g/L/h.The result shows that under the minimum growth pH condition near original strain, the reorganization bacterial strain is significantly increased than original strain acid resistance.Mutant strain can be used for low pH zymotechnique and produce L-lactic acid.Lactic acid concn has improved 42.61% than original strain in the Lc-F34 bacterial strain fermentation liquor, has on average improved 42.7%.
Than under the low condition, the Lc-F34 bacterial strain all is significantly improved than the product acid amount and the volume productive rate of original strain at pH (5.0 and 4.5), illustrates that the acid resistance of reorganization bacterial strain obviously strengthens than original strain.Especially ferment under pH 4.5 conditions, the content of free lactic acid accounts for 20% of total lactic acid content in the fermented liquid, can reduce the consumption of the alkaline matter in the fermenting process, reduces the cost of lactic acid downstream processing; In addition, go for the lactic acid original position separation coupling connection fermentation manufacturing technique (electrodialysis etc.) that new development is got up.
Claims (10)
1, a kind of mutant strain of lactobacillus casei (Lactobacillus casei subsp.rhamnosus)-Lc-F34, it is deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center on September 27th, 2005, deposit number is CGMCC No1466, classification called after: lactobacterium casei rhamnosyl subspecies (Lactobacillus casei subsp.rhamnosus).
2, a kind of method for preparing the described mutant strain of lactobacillus casei of claim 1 (Lactobacillus casei subsp.Rhamnosus)-Lc-F34, it comprises the steps:
The first step, the cultivation of original strain
With original strain-lactobacterium casei rhamnosyl subspecies ATCC11443, adopt the MRS solid medium to carry out separation and purification, colony inoculation bigger on the picking solid plate is in liquid MRS substratum, 37 ℃, 120 rev/mins, concussion overnight incubation, 8000 rev/mins of centrifugal collection thalline, sterilization 0.01M TrisHCl, pH6.8 washed twice are resuspended among sterilization 0.01M TrisHCl, the pH6.8;
Second step, the elementary mutagenesis of bacterial classification
The original strain that adopts two kinds of mutafacient system of ultraviolet ray-UV and nitrosoguanidine-NTG that the first step is cultivated respectively carries out mutagenic treatment, again by low pH flat board, lime carbonate flat board and shake flask test, screening obtains increasing the rate of producing acid of the plant mutant of manying bacterial strain-Lc-UV 1% or more and nitrosoguanidine mutagenesis and acid resistance than the many plant mutant bacterial strain-Lc-NTG of original strain increase more than 1% through the rate of producing acid of ultraviolet mutagenesis and acid resistance than original strain respectively;
The 3rd step, genome reorganization
(1) protoplastis preparation and regeneration
Lc-UV that yeast culture will obtain through UV and the elementary mutagenesis screening of NTG and Lc-NTG mutant strain are in the MRS substratum after the incubated overnight, be inoculated in 30ml respectively and contain 1.0～2.0% glycine liquid MRS substratum, inoculum size is 2～10%, 37 ℃ of shaking culture, 8000 rev/mins of centrifugal collection thalline; The thalline of collecting is washed 2 times with LPB, be resuspended among the LPB, add N,O-Diacetylmuramidase and mutanolysin, final concentration is respectively 1～20mg/mL and 5～50ug/mL, in 37 ℃ of enzymolysis 0.5～2.0h, microscopically is observed the broken wall situation, behind the 90% above thalline broken wall, centrifugal results protoplastis, LPB washing 2 times, be resuspended among the LPB standbyly, the protoplastis number average is 10 in the bacterium liquid
8Cfu/mL;
(2) to pass that nearly formula protoplastis merges be genome reorganization to many female parents
Be divided into two groups after the UV mutagenesis that step (1) is obtained and the protoplastis bacterium liquid equal-volume of NTG mutagenic strain mix, one group places power is that 10～50min is handled in the ultraviolet ray of 15W down, after another group places 60 ℃ of water-baths to handle 30～150min complete inactivation, mix centrifugal, be resuspended among the 50 μ L LPB, add 40% PEG 6000 of 9 times of volumes, room temperature is placed 2～6min, add 5mL LPB dilution, centrifugal, washing are resuspended in 100 μ L LPB, and the multiple dilution is coated with the RM flat board, 37 ℃, CO
2(5%) the incubator lucifuge was cultivated 5 days.The RM flat-plate bacterial colony is for merging the back bacterial strain.Bacterium colony on the RM flat board is washed with sterile saline, and dilution is coated with the pH4.0YE flat board, and 37 ℃, CO
2(5%) the incubator lucifuge is cultivated, and the bacterium colony that obtains on the pH4.0 flat board is washed with physiological saline, suitably dilutes back coating lime carbonate flat board, and 37 ℃, CO
2(5%) incubator is cultivated 48h, and the bacterium colony that transparent circle is bigger on the picking lime carbonate flat board carries out shake-flask culture 17h, measures and produces the acid amount, obtains producing the high mutant strain-Lc-F1 more than 2% of acid molar ratio original strain; A plurality of Lc-F1 bacterial strains continuation steps (1), (2) are carried out protoplastis preparation, deactivation, fusion and pH3.8 flat board, lime carbonate flat board and the shake flask test screening of next round, measure and produce the acid amount, obtain producing the mutant strain-Lc-F2 of acid molar ratio Lc-F1 mutant bacteria plant height more than 2%;
Protoplastis preparation, deactivation, fusion and pH3.6 flat board, lime carbonate flat board and the shake flask test screening of next round are carried out in (1), (2) set by step will to screen the continuation of Lc-F2 bacterial strain once more, measure and produce the acid amount, obtain producing the mutant strain-Lc-F3 of acid molar ratio Lc-F2 mutant bacteria plant height more than 2%;
F3 bacterial strain to above-mentioned acquisition carries out stability test, acid resistance and product acid amount property analysis, obtain the strongest and the fastest mutant strain-Lc-F34 of rate of producing acid of a strain acid resistance, mutant strain of lactobacillus casei promptly of the present invention (Lactobacillus casei subsp.Rhamnosus).
3, the described mutant strain of lactobacillus casei of claim 1 (Lactobacillus casei subsp.Rhamnosus)-Lc-F34 is in the application that is used for anaerobically fermenting production L-lactic acid.
4, mutant strain of lactobacillus casei as claimed in claim 3 (Lactobacillus casei subsp.Rhamnosus)-Lc-F34 is in the application that is used for anaerobically fermenting production L-lactic acid, its processing step is as follows: the pure growth that obtains the Lc-F34 mutant strain through seed enlarged culturing step by step, be inoculated in the sealed fermenting equipment that contains fermention medium by the inoculum size of 2%～20% fermented liq volume concentrations pure growth again the Lc-F34 mutant strain, comprise the primary carbon source of 2%～30% volumetric concentration and the nitrogenous source of 1%～15% volumetric concentration in the fermention medium, all the other are water; Fermented liquid carries out anaerobically fermenting under 35 ℃～50 ℃, 200rpm agitation condition cultivates, and the pH value of control fermented liquid is 3.2～7.0 in the fermenting process, in the fermenting process in the maintenance fermented liquid carbon source volumetric concentration 1%～20%.
5, mutant strain of lactobacillus casei as claimed in claim 4 (Lactobacillus casei subsp.Rhamnosus)-Lc-F34 is characterized in that in the application that is used for anaerobically fermenting production L-lactic acid: the pH value of using alkaline matter control fermented liquid.
6, mutant strain of lactobacillus casei as claimed in claim 4 (Lactobacillus casei subsp.Rhamnosus)-Lc-F34 is in the application that is used for anaerobically fermenting production L-lactic acid, and it is characterized in that: alkaline matter is NaOH, ammoniacal liquor or GaGO
3In one or more.
7, mutant strain of lactobacillus casei as claimed in claim 4 (Lactobacillus casei subsp.Rhamnosus)-Lc-F34 is in the application that is used for anaerobically fermenting production L-lactic acid, it is characterized in that: carbon source is glucose, sucrose, maltose, dextrose syrup or lactose, and nitrogenous source is ammonium sulfate, yeast extract, ammonium nitrate, corn steep liquor, sal epsom, manganous sulfate, Trisodium Citrate, sodium acetate or rice bran.
8, mutant strain of lactobacillus casei as claimed in claim 7 (Lactobacillus casei subsp.Rhamnosus)-Lc-F34 is in the application that is used for anaerobically fermenting production L-lactic acid, and it is characterized in that: carbon source is a glucose; Nitrogenous source is yeast extract or corn steep liquor.
9, mutant strain of lactobacillus casei as claimed in claim 8 (Lactobacillus casei subsp.Rhamnosus)-Lc-F34 is in the application that is used for anaerobically fermenting production L-lactic acid, it is characterized in that: Lc-F34 inoculation amount is 5%～15%, fermenting process control pH is 4.5～6.0, controlled temperature is 37 ℃～42 ℃, carbon source concentration is 8%～20%, and nitrogen concentration-yeast extract is 1.0%～2.5% or corn steep liquor 4.5%～7.0%.
10, mutant strain of lactobacillus casei as claimed in claim 9 (Lactobacillus casei subsp.Rhamnosus)-Lc-F34 is in the application that is used for anaerobically fermenting production L-lactic acid, it is characterized in that: Lc-F34 inoculation amount is 10%, fermenting process pH is 5.5, controlled temperature is 40 ℃, carbon source concentration is 10%～16%, nitrogen concentration-yeast extract be 1.5～2.0% or corn steep liquor be 5.0～6.5%.
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| CN100554405C (en) * | 2007-10-18 | 2009-10-28 | 中国科学院微生物研究所 | A kind of method and special-purpose lactobacillus rhamnosus thereof that produces L-lactic acid |
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| CN102653724A (en) * | 2012-05-30 | 2012-09-05 | 南京工业大学 | Lactobacillus casei and application thereof in fermentation production of L-lactic acid |
| CN102653724B (en) * | 2012-05-30 | 2013-06-12 | 南京工业大学 | Lactobacillus casei and application thereof in fermentation production of L-lactic acid |
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| CN104381932A (en) * | 2014-12-10 | 2015-03-04 | 杭州元佩特生物科技有限公司 | Method for preparing cabbage kimchi rich in nattokinase and pyrroloquinoline quinone |
| CN110643532A (en) * | 2019-09-24 | 2020-01-03 | 扬州大学 | Oil-degraded lactobacillus paracasei and application thereof |
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