CN104031962A - Optimized pEGF gene engineering Lactococcus lactis fermentation medium - Google Patents
Optimized pEGF gene engineering Lactococcus lactis fermentation medium Download PDFInfo
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- CN104031962A CN104031962A CN201410183934.3A CN201410183934A CN104031962A CN 104031962 A CN104031962 A CN 104031962A CN 201410183934 A CN201410183934 A CN 201410183934A CN 104031962 A CN104031962 A CN 104031962A
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Abstract
The invention relates to an optimization of a pEGF gene engineering Lactococcus lactis fermentation medium. The optimization contents comprise a medium component optimization, a fermentation time optimization and an inoculation amount optimization. The components of 1L of an optimized medium contain 6.5g of low protein whey powder, 5g of glucose, 15g of peptone, 1g of a beef extract, 1g of yeast extract powder, 0.1g of vitamin C, 0.25g of magnesium sulfate, 2g of sodium nitrate and 0.4g of ferrous sulfate. The pH value, the optimum fermentation time and the optimum inoculation amount of the optimized fermentation medium are 7.0, 27h and 10% respectively. The thallus concentration of the optimized fermentation medium reaches 2*10<9>CFU/ml, and is obviously higher than that of a basic fermentation medium, so the fermentation effect can be substantially improved. The optimized basic fermentation medium substantially reduces the fermentation cost of pEGF gene engineering Lactococcus lactis.
Description
technical field
The invention belongs to microecology, microorganism fermentation field, more specifically relates to a kind of fermention medium of the pEGF of significantly improving genetically engineered Lactococcus lactis bacteria biomass.
technical background
Pig's epidermal growth factor (porcine epidermal growth factor, pEGF) is the one of Urogastron, is one of the abundantest somatomedin of pig Ruzhong content.Very high at pig colostrum and normal Ruzhong Urogastron concentration content, and its acceptor studies at most in the distribution of chitterlings, and EGF and its receptors bind can be brought into play physiological function.There are some researches show that pig's epidermal growth factor has various biological function, as stimulated the propagation of epidermis and endotheliocyte; Can the growth in stimulating gastrointestinal road and the expression of induction small intestine digestive ferment.For nascent piglet and early-weaned piglets, pEGF can affect the growth of its production performance, immunne response and small intestine, the expression of induction small intestine digestive ferment.In addition, pEGF has elementary female ovarian follicle of stationary phase of promotion and becomes activation ovarian follicle, thereby accelerates the discharge of ovum, therefore has the function that improves mammiferous nest litter size.
Lactococcus lactis is that a class can metabolize sugars, and the bacterium that produces 50% above lactic acid is generally called, and comprises at least 23 genus such as genuslactococcus lactis, lactococcus, genus bifidobacterium, leuconos toc.Itself have the micro-ecology of regulating intestinal canal, promotion nutritive ingredient is digested and assimilated enteron aisle, reduces the cholesterol level in serum, alleviates hypertension and antitumor action.Lactococcus lactis is that facultative anaerobe is the generally acknowledged food-grade microorganisms in the whole world, along with the development of Protocols in Molecular Biology, by gene recombination, it is transformed, there is object, make Lactococcus lactis produce the product of useful heterologous gene selectively, making the healthcare products such as probiotics and live bacterial vaccines or medicine, is an important directions of Lactococcus lactis molecular biology research.
Along with the develop rapidly of genetic engineering technique, we can be by building our needed plasmid vector to the research of the plasmid-encoded characteristic of Lactococcus lactis, by a series of molecular biology methods for example transduce, in conjunction with, electricity transform etc., transferred in Lactococcus lactis, we are expressed by needed goal gene, thereby make genetically engineered Lactococcus lactis integrate thalline, self secretory product, foreign protein.Genetically engineered Lactococcus lactis is applied in feed, can be used as a kind of novel fodder additives, it can produce antimicrobial (especially pathogenic bacterium) material, can also regulate the microecological balance in animal body enteron aisle, strengthen animal body immunizing power, thereby promotion growth of animal, improves food conversion ratio.
summary of the invention
The invention provides a kind of fermentation optimization substratum that can significantly improve pEGF genetically engineered Lactococcus lactis bacteria biomass.
In the present invention, described fermention medium is made up of following component:
1) low albumen whey powder 6.5 ~ 15g/L;
2) dextrose anhydrous 5 ~ 20g/L;
3) peptone 5 ~ 15g/L;
4) extractum carnis 1 ~ 10g/L;
5) yeast extract powder 1 ~ 5g/L;
6) vitamins C 0.1 ~ 0.5g/L;
7) magnesium sulfate 0 ~ 0.25g/L;
8) SODIUMNITRATE 0 ~ 2g/L;
9) ferrous sulfate 0 ~ 0.4g/L;
10) the pH value of described fermentation optimization substratum is 5.8-7.0; The incubation time of described fermentation optimization substratum is 24 ~ 39h; The inoculum size of described fermentation optimization substratum is 2% ~ 10%; Described fermentation optimization training method is cultivated for leaving standstill; Described fermentation culture temperature is 37 DEG C.
In the present invention, in described fermention medium, contain low albumen whey powder 6.5 ~ 15g/L; Preferably, in described fermentation optimization substratum, contain low albumen whey powder 6.5g/L.
In the present invention, in described fermention medium, contain dextrose anhydrous 5 ~ 20g/L; Preferably, in described fermentation optimization substratum, contain dextrose anhydrous 5g/L.
In the present invention, in described fermention medium, contain peptone 5 ~ 15g/L; Preferably, in described fermentation optimization substratum, contain peptone 15g/L.
In the present invention, in described fermention medium, contain extractum carnis 1 ~ 10g/L; Preferably, in described fermentation optimization substratum, contain extractum carnis 1g/L.
In the present invention, in described fermention medium, contain yeast extract powder 1 ~ 5g/L; Preferably, in described fermentation optimization substratum, contain yeast and soak powder 1g/L.
In the present invention, in described fermention medium, contain vitamins C 0.1 ~ 0.5g/L; Preferably, in described fermentation optimization substratum, contain vitamins C 0.1g/L.
The present invention preferably, contains magnesium sulfate 0.25g/L in described fermentation optimization substratum; Described SODIUMNITRATE 2g/L; Described ferrous sulfate 0.4g/L.
Preferably, described fermentation optimization Medium's PH Value is 7.0 in the present invention; Described fermented incubation time is 27h; Described inoculum size is 10%; Described training method is that anaerobism leaves standstill cultivation; Described fermentation culture temperature is 37 DEG C.
More preferably, described fermentation optimization substratum is made up of following component in the present invention: every 1L substratum is containing low albumen whey powder 6.5g, glucose 5g, peptone 15g, extractum carnis 1g, yeast soaks powder 1g, vitamins C 0.1g, magnesium sulfate 0.25g, SODIUMNITRATE 2g, ferrous sulfate 0.4g.
In the present invention, the medium that dissolves described fermentation optimization substratum is this area routine, is preferably water; Described water is described in the routine of this area, as distilled water, distilled water etc.
In the present invention, the preparation method of described fermentation optimization substratum is this area routine, and each component that only need be comprised is simply mixed, then according to the preparation condition preparation of the conventional microbiological culture media in this area; Described conventional preparation condition is 121 DEG C, high pressure moist heat sterilization 15min.
Agents useful for same of the present invention and raw material be commercially available obtaining all.
The positive progressive effect of pEGF genetically engineered Lactococcus lactis fermentation optimization substratum of the present invention is: fermentation optimization substratum of the present invention can provide pEGF genetically engineered Lactococcus lactis well to grow, and has significantly improved the fermentation biomass of pEGF genetically engineered Lactococcus lactis; Meanwhile, fermentation optimization substratum of the present invention greatly reduces the cost of pEGF genetically engineered Lactococcus lactis bacteria fermentation culture medium.
Brief description of the drawings
The growth curve of accompanying drawing 1 pEGF genetically engineered Lactococcus lactis.
Accompanying drawing 2 pEGF expression amounts are measured.
Embodiment
Mode below by experiment embodiment further illustrates the present invention, but does not therefore limit the present invention among described example ranges.
The activation of embodiment 1:pEGF genetically engineered Lactococcus lactis
The preparation of liquid activation medium: M17 substratum 3.76g, dextrose anhydrous 1g, distilled water 100ml, 121 DEG C, high pressure moist heat sterilization, 15min, cooling in super clean bench after, add 100 μ l erythromycin solution, erythromycin strength of solution is 1mg/ml, 4 DEG C save backup.
The preparation of activation medium flat board: M17 substratum 3.76g, dextrose anhydrous 1g, agar powder 15g, distilled water 100ml, 121 DEG C, high pressure moist heat sterilization, 15min, add 100 μ l erythromycin solution, erythromycin strength of solution is 1mg/ml, and every flat board is poured 20-25ml substratum into, in Bechtop after cooled and solidified, sealing, 4 DEG C save backup.
Take under room temperature being stored in bacterial classification in 40% glycerine, the transfering loop picking bacterial classification of crossing with autoclaving in Bechtop, then draws on flat board, and sealing is inverted at 37 DEG C and is cultivated 24 hours.
After 24 hours, with the transfering loop picking list bacterium colony of sterilizing, be inoculated in liquid nutrient medium, described substratum is aforesaid liquid activation medium, leaves standstill at 37 DEG C and cultivates 24 hours.
After 24 hours, pipette 10%(v/v with pipettor) bacterium liquid, be re-seeded in one bottle of liquid activation medium, leave standstill at 37 DEG C and cultivate 24 hours, obtain seed liquor, collect bacterium liquid, 4 DEG C of preservations, for subsequent use.
The mensuration of embodiment 2:pEGF genetically engineered Lactococcus lactis growth curve
By after pEGF genetically engineered Lactococcus lactis activation, with 10%(v/v) inoculum size be inoculated in basic fermention medium, the component of described basic fermention medium is as follows: albumen whey powder 6.5g, glucose 10g, peptone 10g, extractum carnis 5g, yeast soaks powder 2.5g, magnesium sulfate 0.25g, SODIUMNITRATE 1g, sodium-chlor 2g, ferrous sulfate 0.4g, vitamins C 0.3g, distilled water 1000mL, pH7.0.
Liquid amount is the triangle brand dress 80ml substratum of 100ml, at 37 DEG C, leave standstill and cultivate, every 3 hours, adopt blood cell colony counting method to measure the bacterium colony units of different time, taking incubation time as X-coordinate, bacterium colony units is that ordinate zou is drawn growth curve chart (Fig. 1).
The best training sample time of being determined pEGF genetically engineered Lactococcus lactis by growth curve is 27h.
Embodiment 3: adjust the impact of the each component proportions of substratum on pEGF genetically engineered Lactococcus lactis fermentation biomass
In following experiment, adopt two factors, two kinds of medium component ratios of every suboptimization, all the other composition inconvenience, leave standstill at 37 DEG C and cultivate 27h, measure bacterium colony units with blood cell colony counting method.
3.1 other components unchanged, arrange low albumen whey powder concentration 6.5g/L, 10g/L, 15g/L and glucose concn 5g/L, 10g/L, 20g/L, and each combination repeats 3 times, average, (as table 1).Result shows, when low albumen whey powder concentration is 6.5g/L, when glucose concn is 5g/L, bacterium colony units reaches the highest.
Table 1 bacterial biomass lgCFU
3.2 on the basis of 3.1 optimization experiment, and peptone concentration 5g/L, 10g/L, 15g/L and extractum carnis concentration 1g/L, 5g/L, 10g/L are set,
Other components unchanged, each combination repeats 3 times, averages, (as table 2).Result shows, when peptone concentration is 15g/L, when extractum carnis concentration is 1g/L, bacterium colony units reaches the highest.
Table 2 bacterial biomass lgCFU
3.3 on the basis of 3.2 optimization experiment, and yeast is set and soaks powder concentration 1g/L, 2.5g/L, 5g/L and vitamins C 1g/L, 5g/L, 10g/L,
Other components unchanged, each combination repeats 3 times, averages, (as table 3).Result shows, is 1g/L when yeast soaks powder concentration, and when vitamins C concentration is 0.1g/L, bacterium colony units reaches the highest.
Table 3 bacterial biomass lgCFU
The impact of embodiment 4 inoculum sizes on pEGF genetically engineered Lactococcus lactis fermentation biomass
By pEGF genetically engineered Lactococcus lactis respectively with 2%, 5%, 10%, 15%(v/v) inoculum size be inoculated in the fermention medium of having optimized, leave standstill at 37 DEG C and cultivate 27h, measure bacterium colony units (as table 4) with blood cell colony counting method.Result shows, fixes at medium component, and incubation time is constant, and when culture temperature is constant, 10% the little bacterium colony units recording of inoculum size is the highest.
Table 4 bacterial biomass lgCFU
Embodiment 5 pEGF expression amounts are measured
Use fermentation optimization culture medium culturing bacterium, adopt the broken thalline of ultrasonic fragmentation, in thalline after fragmentation, add 8M urea in the ratio of 1:50, because target protein is smaller, carry out protein electrophoresis (plate 2) so adopt Tricine-SDS-Page method, low-concentration sodium hydroxide wash-out target protein, measures target protein content as following table 5 with calibration curve method.
Table 5 pEGF expression amount is measured
Claims (10)
1. a fermentation optimization substratum for pEGF genetically engineered Lactococcus lactis, is characterized in that: the fermention medium after optimization is made up of following component:
1) low albumen whey powder 6.5 ~ 15g/L;
2) dextrose anhydrous 5 ~ 20g/L;
3) peptone 5 ~ 15g/L;
4) extractum carnis 1 ~ 10g/L;
5) yeast extract powder 1 ~ 5g/L;
6) vitamins C 0.1 ~ 0.5g/L;
7) magnesium sulfate 0 ~ 0.25g/L;
8) SODIUMNITRATE 0 ~ 2g/L;
9) ferrous sulfate 0 ~ 0.4g/L;
10) the pH value of described pEGF genetically engineered Lactococcus lactis fermentation optimization substratum is 5.8-7.0; The incubation time of described pEGF genetically engineered Lactococcus lactis fermentation optimization substratum is 24 ~ 39h; The inoculum size of described pEGF genetically engineered Lactococcus lactis fermentation optimization substratum is 2% ~ 10%; Described pEGF genetically engineered Lactococcus lactis fermentation optimization training method is that anaerobism leaves standstill cultivation; Described pEGF genetically engineered Lactococcus lactis fermentation culture temperature is 37 DEG C; In described pEGF genetically engineered lactobacillus-fermented Optimal Medium, add the erythromycin solution of 1ml/L; Described erythromycin strength of solution is 10mg/ml.
2. pEGF genetically engineered Lactococcus lactis fermentation optimization substratum as claimed in claim 1, is characterized in that, contains low albumen whey powder 6.5g/L in described fermentation optimization substratum; Described dextrose anhydrous 5g/L.
3. pEGF genetically engineered Lactococcus lactis fermentation optimization substratum as claimed in claim 2, is characterized in that, in described fermentation optimization substratum, contains peptone 15g/L; Described extractum carnis 1g/L.
4. pEGF genetically engineered Lactococcus lactis fermentation optimization substratum as claimed in claim 3, is characterized in that, in described fermentation optimization substratum, contains yeast extract powder 1g/L; Described vitamins C 0.1g/L.
5. pEGF genetically engineered Lactococcus lactis fermentation optimization substratum as claimed in claim 4, is characterized in that, in described fermentation optimization substratum, contains magnesium sulfate 0.25g/L; Described SODIUMNITRATE 2g/L; Described ferrous sulfate 0.4g/L.
6. pEGF genetically engineered Lactococcus lactis fermentation optimization substratum as claimed in claim 5, it is characterized in that, described fermentation optimization substratum comprises following component: every 1L substratum is containing low albumen whey powder 6.5g, glucose 5g, peptone 15g, extractum carnis 1g, yeast soaks powder 1g, vitamins C 0.1g, magnesium sulfate 0.25g, SODIUMNITRATE 2g, ferrous sulfate 0.4g.
7. pEGF genetically engineered Lactococcus lactis fermentation optimization substratum as claimed in claim 6, is characterized in that, the best fermentation time of described fermentation optimization substratum is 27h; Described fermention medium optimum inoculation amount is 10%; The pH of described fermention medium is 7.0.
8. pEGF genetically engineered Lactococcus lactis fermentation optimization substratum as claimed in claim 7, is characterized in that, adds the erythromycin solution of 100 μ l in described fermentation optimization substratum; Described erythromycin strength of solution is 10mg/ml; Described fermentation optimization substratum liquid amount is the bottled 80ml of 100ml.
9. pEGF genetically engineered Lactococcus lactis fermentation optimization substratum as claimed in claim 8, is characterized in that, described fermentation optimization culture medium culturing mode is cultivated for leaving standstill; Described culture temperature is 37 DEG C.
10. the application of the pEGF genetically engineered Lactococcus lactis fermentation optimization substratum of any one in pEGF genetically engineered Lactococcus lactis fermentation culture as described in claim 1 ~ 9.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107325998A (en) * | 2017-06-20 | 2017-11-07 | 江西嘉博生物工程有限公司 | A kind of Recombinant Lactococcus lactis and construction method for expressing pig's epidermal growth factor gene |
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| CN1560231A (en) * | 2004-02-26 | 2005-01-05 | 天津科技大学 | Preparation process of fermenting agent for beneficial growing lactic bacteria and application in fresh cheese thereof |
| CN101497868A (en) * | 2009-02-26 | 2009-08-05 | 上海应用技术学院 | Improved MRS fluid nutrient medium and use method thereof for screening lactobacillus |
| CN103146738A (en) * | 2013-01-31 | 2013-06-12 | 武汉工业学院 | Construction method and purpose of recombinant lactobacillus acidophilus expressing pig epidermal growth factors |
| CN103710351A (en) * | 2013-12-23 | 2014-04-09 | 江苏众红生物工程创药研究院有限公司 | Recombination lactic acid bacteria including epidermal growth factor and application thereof |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1560231A (en) * | 2004-02-26 | 2005-01-05 | 天津科技大学 | Preparation process of fermenting agent for beneficial growing lactic bacteria and application in fresh cheese thereof |
| CN101497868A (en) * | 2009-02-26 | 2009-08-05 | 上海应用技术学院 | Improved MRS fluid nutrient medium and use method thereof for screening lactobacillus |
| CN103146738A (en) * | 2013-01-31 | 2013-06-12 | 武汉工业学院 | Construction method and purpose of recombinant lactobacillus acidophilus expressing pig epidermal growth factors |
| CN103710351A (en) * | 2013-12-23 | 2014-04-09 | 江苏众红生物工程创药研究院有限公司 | Recombination lactic acid bacteria including epidermal growth factor and application thereof |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107325998A (en) * | 2017-06-20 | 2017-11-07 | 江西嘉博生物工程有限公司 | A kind of Recombinant Lactococcus lactis and construction method for expressing pig's epidermal growth factor gene |
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