CN105368759A - Cultivation method of ketogulonigenium sp - Google Patents
Cultivation method of ketogulonigenium sp Download PDFInfo
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- CN105368759A CN105368759A CN201510995214.1A CN201510995214A CN105368759A CN 105368759 A CN105368759 A CN 105368759A CN 201510995214 A CN201510995214 A CN 201510995214A CN 105368759 A CN105368759 A CN 105368759A
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Abstract
The invention relates to the field of microorganisms, in particular to a cultivation method of ketogulonigenium sp. A new technological process of vitamin C precursor 2-ketone-L-gulonic acid is established, in the process, a brand-new ketogulonigenium sp seed cultivation mode is used, and sorbitol and/or mannitol serve/serves as a carbon source in replacement of sorbose. Under the same cultivation time and cultivation condition, the mycelia content is increased by 1.6 times or more through seed cultivation of sorbitol/mannitol than a sorbose cultivation mode, and high-quality fermentation strains are provided for follow-up fermentation. As for the aspect of the same mycelia content, the highest biomass obtained after 25 hours of cultivation in sorbose can be obtained after 5 hours of cultivation in sorbitol and mannitol. Accordingly, the seed cultivation time is greatly shortened by at least 10 hours.
Description
Technical field
The present invention relates to microorganism field, particularly the cultural method of Ketogulonigenium sp.
Background technology
Vitamins C maintains body growth and the necessary a kind of water-soluble vitamins of metabolism, is widely used in medicine, food, feed and cosmetic field at present, market stability.Current ascorbic main flow working system is two-step fermenting, mainly refers to from D-glucitol, is converted into the process of vitamin C precursor-2-keto-L-gulonic acid through two-step fermentation.The first step, utilizes Gluconobacter oxvdans that D-glucitol is converted into L-sorbose, and second step utilizes the mixed thalline system of raw Ketogulonigenium sp and concomitance bacterium (being mainly genus bacillus) thereof that L-sorbose is converted into 2-keto-L-gulonic acid.Due to this bacterial strain single culture poor growth, affect its industrial application at amplification cultivation, actication of culture, therefore, find suitable culture medium prescription, promote that it grows, have very high value to industrial application.
To the thing Quality Research promoting Ketogulonigenium sp growth, comprise add gsh, tetra-carbonic in tricarboxylic acid cycle or its salt, Thioctic Acid, cofactor all have promoter action to Ketogulonigenium sp growth.Prior art adopts additives matter to attempt improving little bacteria biomass and producing acid amount, but is directly connected to enterprise cost due to the concentration of additives matter, has enterprise's application limitation.Therefore, a kind of cultural method of Ketogulonigenium sp is provided to have important practical significance.
Summary of the invention
In view of this, the invention provides a kind of cultural method cultivating Ketogulonigenium sp.This patent proposition D-glucitol, PEARLITOL 25C replace the Seed Culture protocol of sorbose as carbon source, and the biomass improving bacterial strain reaches 2 times, significant to activated industrial bacterial strain.
In order to realize foregoing invention object, the invention provides following technical scheme:
The invention provides sorbyl alcohol and/or N.F,USP MANNITOL and cultivate the application in Ketogulonigenium sp as carbon source.
In specific embodiments more of the present invention, in described application, sorbyl alcohol or the addition of N.F,USP MANNITOL in the seed culture medium of Ketogulonigenium sp are 15 ~ 45g/L.
Present invention also offers a kind of seed culture medium of Ketogulonigenium sp, replace sorbose as carbon source with sorbyl alcohol and/or N.F,USP MANNITOL.
In specific embodiments more of the present invention, described seed culture medium comprises corn steep liquor 1-5g/L, yeast powder 1 ~ 5g/L, extractum carnis 1 ~ 5g/L peptone 1 ~ 20g/L, urea 0.2 ~ 2g/L, potassium primary phosphate 0.1 ~ 2g/L, magnesium sulfate 0.01 ~ 1g/L, calcium carbonate 0.1 ~ 2g/L, pH is adjusted to 6.5 ~ 7.5,121 DEG C, sterilizing 20min, makes seed culture medium; Solid medium separately adds the agar powder of 20g/L.
Configure sorbyl alcohol and N.F,USP MANNITOL 20g/100ml respectively, 115 DEG C, sterilizing 20min, is added into mass concentration 15 ~ 45g/L in seed culture process.
Present invention also offers a kind of cultural method of Ketogulonigenium sp, select described seed culture medium to carry out seed culture to Ketogulonigenium sp.
In specific embodiments more of the present invention, the Ketogulonigenium sp of activation is inoculated in described seed culture medium by described cultural method, at 30 DEG C, seed culture 48h under the condition of 250r/min.
Present invention also offers a kind of method of fermentative production vitamin C precursor 2-keto-L-gulonic acid, select described seed culture medium to carry out seed culture to Ketogulonigenium sp, collect tunning.
In specific embodiments more of the present invention, the Ketogulonigenium sp of activation is inoculated in described seed culture medium by described method, at 30 DEG C, seed culture 48h under the condition of 250r/min, collects tunning.
Because Ketogulonigenium sp grows separately more weak, find suitable culture medium prescription, promote that it grows, have very high value to industrial application.The present invention compares the effect to Ketogulonigenium sp growth under the glucose of equal in quality concentration, fructose, sorbyl alcohol, sorbose, N.F,USP MANNITOL of this bacterial strain, find under sorbyl alcohol, N.F,USP MANNITOL, cultivate compared with sorbose and have the biomass of 1.6 times, have bacterial classification seed culture and industrial fermentation significant.The present invention relates to biotechnology/field of microbial fermentation, particularly sorbyl alcohol, N.F,USP MANNITOL strengthen strain growth and active application, and analyze its metabolism network by genome, and the change difference of metabolism group to metabolite important in born of the same parents is resolved.Confirm that Ketogulonigenium sp consumes the mechanism that sorbyl alcohol is converted into sorbose, KGA.
The present invention establishes brand-new Ketogulonigenium sp seed culture mode, obtains the new technological flow of vitamin C precursor 2-keto-L-gulonic acid.K.vulgare cultivates the most high-biomass (cell density reaches 3.60) that all reached for 5 hours and cultivate 25 hours in sorbose and be grown on 10 hours in sorbyl alcohol, N.F,USP MANNITOL after in fructose, sorbyl alcohol, N.F,USP MANNITOL, more will significantly better than in sorbose, and most high-biomass (cell density reaches 5.20,4.90) was reached at 25 hours, compare and cultivate and will improve 40.72% and 62.97% respectively in sorbose.Can expect thus, under identical incubation time and culture condition, use sorbyl alcohol/N.F,USP MANNITOL seed culture by than use sorbose training method obtain biomass high 1.5 times and more than, for follow-up fermentation provides the fermented bacterium of more high-quality.If considering from obtaining same bacterial classification amount, in sorbyl alcohol, N.F,USP MANNITOL, cultivating the most high-biomass all having reached for 5 hours and to have cultivated in sorbose 25 hours, greatly shorten the time of seed culture, at least 10 hours.
Accompanying drawing explanation
In order to be illustrated more clearly in the embodiment of the present invention or technical scheme of the prior art, be briefly described to the accompanying drawing used required in embodiment or description of the prior art below.
Fig. 1 shows the growth of Ketogulonigenium sp under different carbon source and base consumption situation; Wherein, A shows the growth curve of Ketogulonigenium sp under different carbon source; B shows the base consumption of Ketogulonigenium sp under different carbon source;
show L-sorbose;
show D-glucitol;
show PEARLITOL 25C;
show D-Glucose;
show D-Fructose;
Fig. 2 shows that Ketogulonigenium sp monose transforms relevant endoenzyme;
Fig. 3 shows that logarithmic phase different carbon source cultivates the content of associated acid product in lower cell; Wherein, Fig. 3 (A) shows L-sorbose; Fig. 3 (B) shows 2-ketone group-L 2-KLG; Fig. 3 (C) shows 2KDG; Fig. 3 (D) shows L-glyconic acid; Fig. 3 (E) shows D-glyconic acid; Fig. 3 (F) shows saccharic acid; Fig. 3 (G) shows mannitic acid;
Fig. 4 shows that the standard substance of different carbon source go out peak position; Wherein, Fig. 4 (A) shows N.F,USP MANNITOL; Fig. 4 (B) shows sorbose; Fig. 4 (C) shows sorbyl alcohol; Fig. 4 (D) shows glucose; Fig. 4 (E) shows fructose.
Embodiment
The invention discloses a kind of cultural method cultivating Ketogulonigenium sp, those skilled in the art can use for reference present disclosure, and suitable improving technique parameter realizes.Special needs to be pointed out is, all similar replacements and change apparent to those skilled in the art, they are all deemed to be included in the present invention.Method of the present invention and application are described by preferred embodiment, related personnel obviously can not depart from content of the present invention, spirit and scope methods and applications as herein described are changed or suitably change with combination, realize and apply the technology of the present invention.
(1) Ketogulonigenium sp seed culture method:
Corn steep liquor 1-5g/L, yeast powder 1-5g/L, extractum carnis 1-5g/L peptone 1-20g/L, urea 0.2-2g/L, potassium primary phosphate 0.1-2g/L, magnesium sulfate 0.01-1g/L, calcium carbonate 0.1-2g/L, pH is adjusted to 6.5-7.5,121 DEG C, sterilizing 20min, makes seed culture medium.Solid medium separately adds the agar powder of 20g/L.
Configure sorbose, sorbyl alcohol, glucose, N.F,USP MANNITOL, fructose 20g/100ml respectively, 115 DEG C, sterilizing 20min, in seed culture process, be added into mass concentration 15 ~ 45g/L.
The Ketogulonigenium sp that solid plate activates is inoculated in substratum and carries out 48 hours seed culture, 30 DEG C, 250r/min, the growth fraction pair under observation different carbon source.
(2) product detects:
Bacteria concentration measures: use 75-6 type ultraviolet spectrophotometer to measure thalli growth curve in culturing process, wavelength adopts 600nm, i.e. OD600.
Base consumption situation measures: high performance liquid chromatography detects, and adopts AminexHPX-87H chromatographic column, Composition distribution.0.5mM sulfuric acid makes moving phase, column temperature 65 DEG C, detector temperature 50 DEG C.
The mark product five kinds of carbon sources being configured to 20 grams per liters carry out high performance liquid chromatography detection acquisition, the peak position of quantitative material and corresponding retention time.Wherein the peak of 10.295min is sorbyl alcohol, and the peak of corresponding retention time 9.427min is sorbose, and the peak of corresponding retention time 10.162min is N.F,USP MANNITOL, and the peak of corresponding retention time 9.184min is glucose, and the peak of corresponding retention time 9.660min is fructose.Obtain the corresponding relation of peak area and mass concentration respectively, fit standard curve, thus, detect the concentration of substrate in Different periods fermented liquid, obtain consumption curve in time.
(3) metabolism group extracting process and analysis
1. born of the same parents' intracellular metabolite thing extracts: get appropriate fermented liquid, be placed in 40% cold methanol solution (-40 DEG C), stops cellular metabolism reaction fast.6000rpm, 4 DEG C of centrifugal 10min.Discard supernatant liquor, add washing cell twice, recentrifuge, discards supernatant liquor.Add water/methyl alcohol (volume ratio 1:1 ,-40 DEG C) the mixed solution 1mL of precooling, and in liquid nitrogen multigelation 3 times to extract born of the same parents' intracellular metabolite thing.The deuterium-labelled succsinic acid of 50 μ L0.040mg/mL is added as interior mark, vacuum-drying in 200 μ L extracts.
2. derivatize: will be dissolved in after sample lyophilize in 50 μ L20mg/mL methoxy amine hydrochlorate/pyridine solutions, 40 DEG C of 90min carry out oximation reaction; Add 80 μ LN-methyl-N-(trimethyl silane) trifluoroacetamides again, 37 DEG C of 30min carry out trimethyl silicone hydride reaction, obtain the born of the same parents' intracellular metabolite thing through derivatize.
3. the detection of metabolite: use GC-TOF-MS to measure, chromatographic condition used and the concrete chromatographic condition of mass spectrometry parameters are: 1 μ L sample, according to the splitting ratio sample introduction of 1:10, is separated through DB-5MS capillary chromatograph; Column oven temperature programming after sample introduction, according to first 70 DEG C of 2min, again with 8 DEG C of min
-1rise to 290 DEG C of 3min; High-purity helium is carrier gas, and keep constant pressure 91Kpa, injection port and Link Port temperature are 280 DEG C.Concrete Mass Spectrometry Conditions is: electricity bombardment ionization (EI) ionizes mode; Source temperature is 250 DEG C, and electronics bombarding energy is 70eV, and transmitter current is 40 μ A; The m/z mass range obtained is between 50-800.
4. the qualitative analysis of metabolite: application Masslynx4.1 software detects mass spectra peak, again with commercialization database NIST (NationalInstituteofStandardsandTechnologymassspectrallib rary, 2011) compare, determine the chemical composition of born of the same parents' intracellular metabolite thing.
The present invention is that the seed culture mode of substrate improves and reaches 1.6 times with sorbose to the more current industrial application of Ketogulonigenium sp biomass, greatly can increase the inoculum size of subsequent fermentation, 10 hours seed fermentation time can be shortened, reduce inoculation culture amount 30%, thus reduce power consumption 20%, enhance productivity more than 20%, liberation equipment and manpower.
The invention provides raw materials used and reagent in the cultural method cultivating Ketogulonigenium sp all to be buied by market.
Below in conjunction with embodiment, set forth the present invention further:
Embodiment 1
To add the seed culture basal growth of sorbose in contrast, select to have with sorbose monose in the sorbyl alcohol of close molecular mass, N.F,USP MANNITOL, fructose, glucose 4 to screen and grow more useful carbon source to it, 4 kinds of carbon sources are all comparatively easier to the raw material of acquisition in industrial-scale application, wherein, sorbyl alcohol is as the precursor substance of sorbose, and price is more cheap.K.vulgare cultivates 48h observation growth and substrate transforms in time, as shown in the figure.K.vulgare grows in sorbyl alcohol, N.F,USP MANNITOL will significantly better than in sorbose, compares and cultivates and will improve 40.72% and 62.97% respectively in sorbose, and this may be transported in thalline with them, and to participate in polystep reaction relevant.By high performance liquid chromatography obtain to five kinds of substrates in time wear rate curve as shown in Figure 1.Raw data refers to table 1 ~ table 10:
The raw data of Figure 1A:
The growth curve of table 1 Ketogulonigenium sp under the seed culture medium adding sorbose
| Time | Sorbose-1 | Sorbose-2 | On average | Error line |
| 0 | 1.458 | 1.518 | 1.488 | 0.0424 |
| 5 | 2.634 | 2.658 | 2.646 | 0.017 |
| 10 | 3.168 | 3.144 | 3.156 | 0.017 |
| 15 | 3.126 | 3.21 | 3.168 | 0.0594 |
| 20 | 3.282 | 3.186 | 3.234 | 0.0679 |
| 25 | 3.606 | 3.564 | 3.585 | 0.0297 |
The growth curve of table 2 Ketogulonigenium sp under the seed culture medium adding sorbyl alcohol
| Time | Sorbyl alcohol-1 | Sorbyl alcohol-2 | On average | Error line |
| 0 | 1.446 | 1.434 | 1.44 | 0.00849 |
| 5 | 3.576 | 3.582 | 3.579 | 0.00424 |
| 10 | 5.004 | 5.07 | 5.037 | 0.04667 |
| 15 | 5.178 | 5.148 | 5.163 | 0.02121 |
| 20 | 4.746 | 4.92 | 4.833 | 0.12304 |
| 25 | 5.208 | 4.932 | 5.07 | 0.19516 |
The growth curve of table 3 Ketogulonigenium sp under the seed culture medium adding N.F,USP MANNITOL
| Time | N.F,USP MANNITOL-1 | N.F,USP MANNITOL-2 | On average | Error line |
| 0 | 1.38 | 1.416 | 1.398 | 0.02546 |
| 5 | 3.282 | 3.282 | 3.282 | 0 |
| 10 | 4.716 | 4.74 | 4.728 | 0.01697 |
| 15 | 4.494 | 4.422 | 4.458 | 0.05091 |
| 20 | 4.164 | 4.08 | 4.122 | 0.0594 |
| 25 | 4.902 | 4.782 | 4.842 | 0.08485 |
The growth curve of table 4 Ketogulonigenium sp under the seed culture medium adding glucose
| Time | Glucose-1 | Glucose-2 | On average | Error line |
| 0 | 1.404 | 1.404 | 1.4 | 0 |
| 5 | 2.826 | 2.826 | 2.83 | 0 |
| 10 | 3.582 | 3.678 | 3.63 | 0.06788 |
| 15 | 3.618 | 3.678 | 3.65 | 0.04243 |
| 20 | 3.474 | 3.486 | 3.48 | 0.00849 |
| 25 | 3.768 | 3.918 | 3.84 | 0.10607 |
The growth curve of table 5 Ketogulonigenium sp under the seed culture medium adding fructose
| Time | Fructose-1 | Fructose-2 | On average | Error line |
| 0 | 1.386 | 1.374 | 1.38 | 0.0085 |
| 5 | 3.366 | 3.36 | 3.363 | 0.0042 |
| 10 | 3.432 | 3.498 | 3.465 | 0.0467 |
| 15 | 3.234 | 3.348 | 3.291 | 0.0806 |
| 20 | 3.468 | 3.372 | 3.42 | 0.0679 |
| 25 | 3.492 | 3.486 | 3.489 | 0.0042 |
The raw data of Figure 1B is in table 6 ~ table 10.
Sorbose concentration curve over time in table 6 substratum
| Time | Sorbose-1 | Sorbose-2 | Sorbose-1 | Sorbose-2 | On average | Error line |
| 0 | 364095 | 366426 | 18.43162589 | 18.5587 | 18.49516 | 0.089857 |
| 5 | 232139 | 233757 | 11.23789742 | 11.3261 | 11.282 | 0.062372 |
| 10 | 220443 | 218460 | 10.60027694 | 10.49217 | 10.54622 | 0.076442 |
| 15 | 212737 | 215521 | 10.18017576 | 10.33195 | 10.25606 | 0.10732 |
| 20 | 204424 | 210339 | 9.726983296 | 10.04945 | 9.888215 | 0.228016 |
| 25 | 200434 | 204644 | 9.509463998 | 9.738977 | 9.62422 | 0.16229 |
Sorbitol concentration curve over time in table 7 substratum
| Time | Sorbyl alcohol-1 | Sorbyl alcohol-2 | Sorbyl alcohol-1 | Sorbyl alcohol-2 | On average | Error line |
| 0 | 365228 | 357987 | 19.3895215 | 19.010602 | 19.20006175 | 0.2679365 |
| 5 | 256800 | 262082 | 13.71551472 | 13.99192029 | 13.8537175 | 0.1954483 |
| 10 | 194875 | 200994 | 10.47499686 | 10.79520241 | 10.63509964 | 0.2264195 |
| 15 | 171625 | 180361 | 9.258330891 | 9.715483317 | 9.486907104 | 0.3232556 |
| 20 | 161205 | 167462 | 8.713055218 | 9.040482271 | 8.876768745 | 0.2315259 |
| 25 | 156399 | 150899 | 8.461558588 | 8.173745133 | 8.317651861 | 0.2035148 |
Mannitol concentration curve over time in table 8 substratum
| Time | N.F,USP MANNITOL-1 | N.F,USP MANNITOL-2 | N.F,USP MANNITOL-1 | N.F,USP MANNITOL-2 | On average | Error line |
| 0 | 389803 | 388300 | 20.711 | 20.6314353 | 20.67127 | 0.05634 |
| 5 | 335140 | 345096 | 17.813 | 18.341244 | 18.07737 | 0.37318 |
| 10 | 237418 | 239092 | 12.633 | 12.7221015 | 12.67773 | 0.06275 |
| 15 | 199696 | 200548 | 10.634 | 10.6789311 | 10.65635 | 0.03194 |
| 20 | 183407 | 182120 | 9.7703 | 9.70208537 | 9.736197 | 0.04824 |
| 25 | 166748 | 2E+05 | 8.8872 | 8.53424897 | 8.710742 | 0.2496 |
Glucose concn curve over time in table 9 substratum
| Time | Glucose-1 | Glucose-2 | Glucose-1 | Glucose-2 | On average | Error line |
| 0 | 344923 | 344501 | 20.22238718 | 20.19752563 | 20.2099564 | 0.017579773 |
| 5 | 248635 | 256005 | 14.54971132 | 14.9839048 | 14.76680806 | 0.307021149 |
| 10 | 196462 | 202442 | 11.47601037 | 11.82831389 | 11.65216213 | 0.24911621 |
| 15 | 169651 | 170405 | 9.896476965 | 9.940897844 | 9.918687404 | 0.031410305 |
| 20 | 154441 | 170444 | 9.000400613 | 9.943195475 | 9.471798044 | 0.666656641 |
| 25 | 160433 | 164696 | 9.353411099 | 9.604559915 | 9.478985507 | 0.177589031 |
Fructose concentration curve over time in table 10 substratum
| Time | Fructose-1 | Fructose-2 | Fructose-1 | Fructose-2 | On average | Error line |
| 0 | 351588 | 348471 | 19.26425026 | 19.09751583 | 19.180883 | 0.117899041 |
| 5 | 233748 | 250519 | 12.9607583 | 13.85787188 | 13.409315 | 0.634355092 |
| 10 | 224508 | 231364 | 12.46649264 | 12.83323348 | 12.649863 | 0.259324936 |
| 15 | 219242 | 224817 | 12.18480401 | 12.48302165 | 12.333913 | 0.210871721 |
| 20 | 215733 | 219857 | 11.99710074 | 12.21770156 | 12.107401 | 0.155988337 |
| 25 | 223264 | 217049 | 12.39994865 | 12.06749615 | 12.233722 | 0.235079417 |
Select sorbose, sorbyl alcohol, N.F,USP MANNITOL, fructose, glucose, K.vulgare cultivates 48h observation growth and substrate transforms in time, as shown in Figure 1.From 1A, the growth of K.vulgare in sorbyl alcohol, N.F,USP MANNITOL, fructose, glucose is comparatively all significantly improved for 5 hours in sorbose, especially in fructose, sorbyl alcohol, N.F,USP MANNITOL, most high-biomass (cell density reaches 3.60) that to cultivate 25 hours in sorbose has all been reached and be grown on 10 hours in sorbyl alcohol, N.F,USP MANNITOL after, more will significantly better than in sorbose, and most high-biomass (cell density reaches 5.20,4.90) was reached at 25 hours, compare and cultivate and will improve 40.72% and 62.97% respectively in sorbose.Can expect thus, under identical incubation time and culture condition, use sorbyl alcohol/N.F,USP MANNITOL seed culture by than use sorbose training method obtain biomass high 1.5 times and more than, for follow-up fermentation provides the fermented bacterium of more high-quality.If considering from obtaining same bacterial classification amount, in sorbyl alcohol, N.F,USP MANNITOL, cultivating the most high-biomass all having reached for 5 hours and to have cultivated in sorbose 25 hours, greatly shorten the time of seed culture, at least 10 hours.Changed in time from 1B, the K.vulgare concentration of substrate in sorbose, sorbyl alcohol, N.F,USP MANNITOL, fructose, glucose, all reach plateau in the 15-20 little time, illustrate that thalline no longer consumes substrate.By plots changes, find out that thalline had a large amount of absorption substrates at 0-5 hour, for production capacity and metabolism growth, later stage 5-10 hour, cell under only having N.F,USP MANNITOL to cultivate has a large amount of phenomenon consuming substrate, and its biomass also has obvious amplification in the 5-10 little time.Illustrate that bacterium connects bacterium amount identical, under same culture conditions, be about 50%, and the maximum biomass of its plateau has obvious difference to the consumption of substrate generally.In industrial application, the price outline of sorbyl alcohol/N.F,USP MANNITOL lower than sorbose, and when required consumption is identical, has the biomass of 60% to improve, and has very high value to reduction production cost.
Embodiment 2
By two generation sequencing technologies obtain genome sequencing result to this bacterial strain, obtain a karyomit(e) and two plasmids, its sequence is delivered on the website of US National Biotechnology Information center NCBI (NationalCenterforBiotechnologyInformation), obtain numbering CP012908, CP012909 and CP012910.Obtain the main metabolic pathway to substrate utilization by KEGGserver on-line analysis software, find the carbohydrate key transformation path famine producing Ketogulonigenium sp.May realize mainly through its a large amount of cross-film apodehydrogenase the utilization of different monose.
Embodiment 3
In order in further observation of cell to the metabotic change after different substrate utilization, obtain the changing conditions of logarithmic phase (12 hours) born of the same parents' intracellular metabolite thing by metabonomic analysis means, obtain the relative different of different acid product.Wherein, sorbose and KGA is detected when sorbyl alcohol is substrate.Illustrate that this bacterial strain has the mechanism that metabolism sorbyl alcohol produces acid equally.Mannitic acid is detected when N.F,USP MANNITOL is substrate.In addition, under five kinds of substrates, product 2KDG, L-glyconic acid, saccharic acid, the obvious difference of D-glyconic acid, relevant from different metabolic waies.Raw data is not as table 11 ~ 15 (relative quantity has unit):
Table 11 logarithmic phase is added sorbose and is cultivated lower intracellular different product assay
Table 12 logarithmic phase is added sorbyl alcohol and is cultivated lower intracellular different product assay
Table 13 logarithmic phase is added N.F,USP MANNITOL and is cultivated lower intracellular different product assay
Table 14 logarithmic phase is added glucose and is cultivated lower intracellular different product assay
Table 15 logarithmic phase is added fructose and is cultivated lower intracellular different product assay
Wherein, the accumulating amount of KGA in sorbose cultured cells is the highest, because its acid toxicity may have restriction to the growth of thalline.In addition, the accumulating amount of 2KDG also in the culturing cell of sorbose is maximum, in sorbyl alcohol cultured cells, comparatively has the height of 25 times, also may there is toxicity to the growth of Ketogulonigenium sp.And glyconic acid in the culturing cell of sorbyl alcohol, saccharic acid, mannitic acid are obvious compared with the accumulating amount in N.F,USP MANNITOL lower, are that its growth is more better than the reason in N.F,USP MANNITOL.
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (8)
1. sorbyl alcohol and/or N.F,USP MANNITOL are cultivating the application in Ketogulonigenium sp as carbon source.
2. application according to claim 1, is characterized in that, sorbyl alcohol or the addition of N.F,USP MANNITOL in the seed culture medium of Ketogulonigenium sp are 15 ~ 45g/L.
3. a seed culture medium for Ketogulonigenium sp, is characterized in that, replaces sorbose as carbon source with sorbyl alcohol and/or N.F,USP MANNITOL.
4. seed culture medium according to claim 3, is characterized in that, comprises corn steep liquor 1-5g/L, yeast powder 1 ~ 5g/L, extractum carnis 1 ~ 5g/L peptone 1 ~ 20g/L, urea 0.2 ~ 2g/L, potassium primary phosphate 0.1 ~ 2g/L, magnesium sulfate 0.01 ~ 1g/L, calcium carbonate 0.1 ~ 2g/L, pH is adjusted to 6.5 ~ 7.5,121 DEG C, sterilizing 20min, makes seed culture medium; Solid medium separately adds the agar powder of 20g/L;
Configure sorbyl alcohol and N.F,USP MANNITOL 20g/100ml respectively, 115 DEG C, sterilizing 20min, adds mass concentration 15 ~ 45g/L in seed culture process.
5. a cultural method for Ketogulonigenium sp, is characterized in that, selects the seed culture medium as described in claim 3 or 4 to carry out seed culture to Ketogulonigenium sp.
6. cultural method according to claim 5, is characterized in that, is inoculated in by the Ketogulonigenium sp of activation in the seed culture medium as described in claim 3 or 4, at 30 DEG C, seed culture 48h under the condition of 250r/min.
7. a method for fermentative production vitamin C precursor 2-keto-L-gulonic acid, is characterized in that, selects the seed culture medium as described in claim 3 or 4 to carry out seed culture to Ketogulonigenium sp, collects tunning.
8. method according to claim 7, is characterized in that, is inoculated in by the Ketogulonigenium sp of activation in the seed culture medium as described in claim 3 or 4, at 30 DEG C, seed culture 48h under the condition of 250r/min, collects tunning.
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| CN101845475A (en) * | 2010-04-30 | 2010-09-29 | 仪宏 | Nutrition-enhanced culture medium for preparing 2-KGA through fermentation and method thereof for preparing 2-KGA |
| CN104357529A (en) * | 2014-10-15 | 2015-02-18 | 沈阳药科大学 | Method for improving production capacity of 2-KGA (2-keto-L-gulonic acid) through enhancement of Ketogulonogeniumvulgarum carbon metabolism level |
| CN104404121A (en) * | 2014-12-10 | 2015-03-11 | 天津大学 | Method for fermentation production of 2-keto-L-ulonic acid |
| CN105112488A (en) * | 2015-10-22 | 2015-12-02 | 天津大学 | Fermenting production method of 2-ketone-L-gulconic acid |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101845475A (en) * | 2010-04-30 | 2010-09-29 | 仪宏 | Nutrition-enhanced culture medium for preparing 2-KGA through fermentation and method thereof for preparing 2-KGA |
| CN104357529A (en) * | 2014-10-15 | 2015-02-18 | 沈阳药科大学 | Method for improving production capacity of 2-KGA (2-keto-L-gulonic acid) through enhancement of Ketogulonogeniumvulgarum carbon metabolism level |
| CN104404121A (en) * | 2014-12-10 | 2015-03-11 | 天津大学 | Method for fermentation production of 2-keto-L-ulonic acid |
| CN105112488A (en) * | 2015-10-22 | 2015-12-02 | 天津大学 | Fermenting production method of 2-ketone-L-gulconic acid |
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