CN103305566A - Culture method for improving yield and viscosity of bacterial polysaccharides - Google Patents
Culture method for improving yield and viscosity of bacterial polysaccharides Download PDFInfo
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- CN103305566A CN103305566A CN2013102379978A CN201310237997A CN103305566A CN 103305566 A CN103305566 A CN 103305566A CN 2013102379978 A CN2013102379978 A CN 2013102379978A CN 201310237997 A CN201310237997 A CN 201310237997A CN 103305566 A CN103305566 A CN 103305566A
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Abstract
The invention provides a culture method for improving yield and viscosity of bacterial polysaccharides. By using the characteristic that penbritin can be simultaneously used as a mutagenic agent and bacterial cell wall peptidoglycan to synthesize stress factors, the penbritin is added to subsequent subculture processes of bacterial isolates for producing different polysaccharides; the adding amount of the penbritin is gradually increased along with an increase of passage number, so as to improve thrill on bacterial cells; and a series of high-producing strains with different viscosities and improvement degrees can be screened in the continuous passage process, so as to adapt to different application requirements. Therefore, the production cost is reduced.
Description
(1) technical field
The present invention relates to a kind of cultural method that improves bacterial polysaccharides output and viscosity.
(2) background technology
Bacterial polysaccharides is by bacterial cell secretion, the polymer that is coupled together through condensation, dehydration by a plurality of monosaccharide units or derivative.Bacterial polysaccharides mainly is present in occurring in nature with three kinds of forms: stick on the cell surface, be secreted in the substratum or the composition cellular component.According to the difference of repeating unit, polysaccharide generally can be divided into homopolysaccharide and mixed polysaccharide, and the former only contains a kind of monose, and the latter is contained two or more monose.Bacterial polysaccharides is that fermentation using bacteria produces, and therefore compares with the animals and plants polysaccharide, and its production is subjected to the influence of factors such as geographical environments such as season, region and disease and pest, weather, natural disaster less, with short production cycle, and output and quality are all very stable, and cost performance is higher.Bacterial polysaccharides has particular structure and good physicochemical property simultaneously, and therefore, in medicine, food, chemical industry, oil production, environment protection and other fields have widely uses.
Among all various bacterium exocellular polysaccharides, gelling gum and xanthan gum are two kinds of the most successful polysaccharide of business development.Xanthan gum is the exocellular polysaccharide that is produced by xanthomonas campestris (Xanthomnas campestris) fermentation.Its structure is made up of the β-glucose main chain of (1 → 4) key connection and the side chain of trisaccharide unit; Side chain is made up of seminose, glucuronic acid and seminose.The seminose that connects main chain is acetylation, and the hydroxyl of terminal seminose and the carbonyl of pyruvic acid form acetal.Gelling gum is the exocellular polysaccharide that is produced through aerobic fermentation by Sphingol single-cell (Sphingomonas elodea).The linear anionic polysaccharide of being formed by 3 kinds of monose repeated polymerization; that is: 2 molecule D-glucose; the L-rhamnosyl of 1 molecule D-glucuronic acid and 1 molecule, in its natural structure, the C2 of first glucose molecule and C6 are replaced by ethanoyl and glyceroyl respectively.Xanthan gum and gelling gum are successively allowed can be used as the foodstuff additive use by U.S. FDA and other a plurality of countries.
But because the output that current most polysaccharide produces bacterium is relatively low, production cost is higher, and its viscosity is lower, has influenced its using value to a certain extent.Current research focus is to increase output by the whole bag of tricks to improve using value.Many investigators have improved polysaccharide yield by the transformation to polysaccharide biosynthetic pathway genes involved.But because gelling gum and xanthan gum etc. as foodstuff additive, are genetic engineering modified bacterial strain if it produces bacterial strain, can cause that the human consumer is to the worry of its security.And the genes involved of the biosynthetic pathway of some polysaccharide is not illustrated, and can't transform it by genetic engineering means.Also have research to handle producing bacterial strain with physics or chemical mutagen, but because these type of mutagenic compound are bigger to cell injury, the positive variation rate that therefore obtains high yield high viscosity bacterial strain is lower, screening efficiency is low.Therefore, press for a kind of high efficiency method with non-genomic engineering of ubiquity, transform producing bacterial strain, screening can be produced has higher output yield and more full-bodied polysaccharide production bacterial strain.
Many cells can produce active oxygen under the effect of microbiotic such as penbritin, thereby damage dna produces sudden change.Therefore, penbritin also can be considered to a kind of relatively mild mutagenic compound.In addition, form though various bacterial polysaccharides has various structure and monose, except a few polysaccharide, the first step in the biosynthetic pathway of other most of bacterium exocellular polysaccharides and bacteria cell wall peptidoglycan have a similarity.The synthetic the first step of exocellular polysaccharide be will activation nucleosides sugar (NDP-sugars) be connected with fat carrier on the cytolemma, other nucleosides sugar of addition is progressively finished the assembling of repeating unit then.This biosynthetic process is synthetic similar to bacteria cell wall moiety peptidoglycan.And the building-up process of peptidoglycan can be suppressed by the microbiotic penbritin.In order to breed under the inhibition of penbritin, cell need synthesize the nucleosides sugar of more fat carrier and activation.Therefore the cell that penbritin is had resistance, the nucleosides sugar synthesis capability that may have stronger fat carrier and activation, therefore under the condition that antibiotic-free exists, these many synthetic substrates also can be used to synthetic polysaccharide, have promoted the synthetic performance of polysaccharide of amicillin resistance bacterial strain.Therefore, penbritin can be used as a kind of mutagenic compound, promotes cell to produce sudden change, also is simultaneously a kind of factor of coercing that promotes the synthetic more the more sugar of cell.
(3) summary of the invention
The objective of the invention is to utilize the penbritin can be simultaneously as mutagenic compound and the synthetic characteristic of coercing the factor of bacteria cell wall peptidoglycan, a kind of cultural method that improves different bacterium polysaccharide yield and viscosity is provided, thereby bacterial polysaccharides generation bacterium was progressively improved the content raising of penbritin to the stimulation of bacterial cell during continuous passage was cultivated, and further can be used for screening the full-bodied bacterial polysaccharides of high yield and produced bacterium.
The technical solution used in the present invention is:
A kind of cultural method that improves bacterial polysaccharides output and viscosity, described method comprises:
(1) chooses the bacterium conduct bacterial strain to be cultivated that produces exocellular polysaccharide, measure penbritin to the minimal inhibitory concentration of this bacterial strain, be designated as MIC;
(2) will wait to cultivate inoculation to the appropriate liquid substratum, add the concentration penbritin of 1/3～2/3 concentration of the corresponding MIC of bacterial strain for this reason, cultivate 24h in 30 ℃, 200rpm, the gained nutrient solution is transferred and is cultivated under the same terms in the liquid nutrient medium that adds the same concentrations penbritin with the inoculum size of 10% volume ratio; Described liquid nutrient medium is the conventional liquid nutrient medium that is applicable to the cultivation product extracellular polysaccharide strains that goes down to posterity;
(3) operation of repeating step (2), corotation connects and cultivates 5 times under this penbritin concentration;
(4) improve penbritin concentration 5～20 μ g/ml on step (2) basis, according to step (2) method, corotation connects and cultivates 5 times under this penbritin concentration;
(5) improve penbritin concentration 5～20 μ g/ml on step (4) basis, according to step (2) method, corotation connects and cultivates 5 times under this penbritin concentration;
(6) operation of repeating step (5) improves constantly the cultivation of transferring of penbritin concentration, and corotation connects to be cultivated 100～200 times.When being used for bacterial strain screening, can connect 30～50 end of taking turns and transfer at revolution, get nutrient solution, gradient dilution (adds 15g/L agar) again in the YM liquid nutrient medium in the YM flat board that contains same concentration penbritin, random choose list bacterium colony, fermentation, and measure its viscosity and polysaccharide yield.
Preferably, described liquid nutrient medium (YM liquid nutrient medium) composed as follows: peptone 5g/L, Fructus Hordei Germinatus soak powder 3g/L, glucose 10g/L, and yeast powder 3g/L, solvent are water, pH7.0.
Preferably, the bacterium of described product exocellular polysaccharide is xanthomonas campestris or Sphingol single-cell.
Beneficial effect of the present invention is mainly reflected in: utilize method provided by the invention, in the continuous passage process of producing exocellular polysaccharide, add penbritin, and progressively improve its content, utilize penbritin to can be used as gentle mutagenic compound and coerce the dual nature of the factor, improve various bacteria exopolysaccharides and viscosity.Penbritin of the present invention is coerced the continuous passage method, handle the bacterium of various product exocellular polysaccharides, especially under the situation of not knowing its biosynthetic pathway genes involved, improve polysaccharide yield and viscosity, and can avoid the human consumer to genetic engineering means the worry of the harm that may bring, be the method that a kind of general bacterium exocellular polysaccharide content and viscosity improve.Simultaneously, in the different treatment stage, a series of different strains that polysaccharide viscosity increases gradually that produce can be obtained, different application requiring can be adapted to.
(4) embodiment
Below with xanthan gum produce bacterium xanthomonas campestris and gelling gum produce the bacterium Sphingol single-cell be example the present invention is described further in conjunction with specific embodiments, but protection scope of the present invention is not limited in this:
Embodiment 1: penbritin produces the mensuration of bacterium xanthomonas campestris and gelling gum generation bacterium Sphingol single-cell minimal inhibitory concentration to xanthan gum
1, xanthomonas campestris Xanthomonas campestris ATCC 13951(is available from ATCC) and Sphingol single-cell Sphingomonas elodea ATCC 31461(available from ATCC) be inoculated in the YM liquid nutrient medium, 30 ℃, 200rpm is cultured to logarithmic phase;
2, nutrient solution is switched to the 1:1000 volume ratio on 96 orifice plates that contain different concns penbritin (10～200 μ g/ml), cultivates 24h for 30 ℃;
3, measure the 0h that cultivates and the optical density value OD that cultivates the 24h that finishes with microplate reader respectively
595
4, the lowest drug concentration with complete bacteria growing inhibiting in aperture is minimal inhibitory concentration;
5, after measured, penbritin is respectively 85 μ g/ml and 90 μ g/ml to the minimal inhibitory concentration of xanthomonas campestris Xanthomonas campestris ATCC 13951 and Yi Le Sphingol single-cell Sphingomonas elodea ATCC 31461.
Embodiment 2: xanthomonas campestris and Sphingol single-cell penbritin are coerced the mutagenesis continuous passage and are cultivated
1, xanthomonas campestris ATCC 13951 and Yi Le Sphingol single-cell ATCC 31461 are inoculated in the YM liquid nutrient medium, and 30 ℃, 200rpm cultivates 24h; The YM liquid nutrient medium is formed: peptone 5g/L, Fructus Hordei Germinatus soak powder 3g/L, glucose 10g/L, and yeast powder 3g/L, solvent are water, pH7.0;
2, respectively above-mentioned culture is transferred respectively in the YM liquid nutrient medium that contains 50 μ g/ml penbritins with 10% inoculum size, 30 ℃, 200rpm cultivates 24h;
3, continue to transfer respectively in the YM liquid nutrient medium that contains 50 μ g/ml penbritins with 10% inoculum size, 30 ℃, 200rpm cultivates 24h; Repeat this program, cell is gone down to posterity 5 times in the YM liquid nutrient medium of 50 μ g/ml penbritins;
4, xanthomonas campestris and Sphingol single-cell are cultivated in the YM of the penbritin that contains 50 μ g/ml liquid nutrient medium and are gone down to posterity after 5 times, and the content of penbritin improves 10 μ g/ml, to total concn be 60 μ g/ml, 30 ℃, 200rpm cultivates 24h;
5, same penbritin concentration (60 μ g/ml), the same terms goes down to posterity 5 times, continues to improve concentration to the 70 μ g/ml of penbritin, repeats to improve after 5 times the concentration of penbritin again.By that analogy, until the tolerance concentration of maximum.In this experiment, final xanthomonas campestris and sphingosine can tolerate the penbritin of 350 μ g/ml.
Embodiment 3: penbritin is coerced the screening of mutant strain in the mutagenesis continuous passage culturing process
1, draws go down to posterity 55 times (penbritin concentration is 150 μ g/ml) respectively, 105 times (penbritin concentration is 250 μ g/ml), and xanthomonas campestris and the Sphingol single-cell nutrient solution of 155 times (penbritin concentration is 350 μ g/ml), gradient dilution is coated in the YM solid plate that contains corresponding penbritin concentration, cultivates 72h for 30 ℃;
2,20 single bacterium colonies of random choose from the culture dish of three different concns of xanthomonas campestris and Sphingol single-cell respectively, be transferred in the inclined-plane that contains YM solid medium (adding 15g/L agar in the YM liquid nutrient medium again), cultivate 72h for 30 ℃, stand-by.
Embodiment 4: the mutant strain leavening property is measured
1, the xanthomonas campestris ATCC 13951 of above-mentioned slant preservation and Yi Le Sphingol single-cell ATCC 31461 are seeded in the YM liquid nutrient medium, cultivate 72h for 30 ℃;
2, be forwarded in separately the fermention medium with 10% inoculum size, cultivate 48h for 30 ℃, wherein the fermention medium of xanthomonas campestris consists of: glucose 30g/L, yeast powder 3g/L, K
2HPO
42g/L, MgSO
40.1g/L, KH
2PO
42g/L, solvent are water, pH7.0; The fermention medium of Sphingol single-cell is: sucrose 30g/L, yeast powder 0.2g/L, soybean protein powder 2g/L, K
2HPO
42g/L, MgSO
40.6g/L, KH
2PO
42g/L, solvent are water, pH7.0; 3, fermented sample is measured polysaccharide yield and fermentation broth viscosity respectively, and its measurement result sees the following form:
Table 1: the mutant strain leavening property relatively after xanthomonas campestris and Sphingol single-cell wild type strain and penbritin were coerced mutagenesis
a 
aStrain number such as SE150G55-1 and XC150G55-1 etc., wherein SE represents Yi Le Sphingol single-cell mutant strain, XC represents the xanthomonas campestris mutant strain, behind the SE 150,250,350 representative is gone down to posterity respectively that final penbritin concentration is respectively 150 in the process, 250, it is 55,105 and 155 times that 350 μ g/ml, G55, G105 and G155 represent the number of times that goes down to posterity respectively ,-1～-20 the expression be different single bacterium colony of 20 random chooses.
4, as can be seen from the table, go down to posterity after 55 times, it is 150 μ g/ml that penbritin is coerced concentration, though it is not very obvious separating the raising of the polysaccharide yield that obtains mutant strain (gelling gum produces bacterium and xanthan gum produces bacterium) this moment at random, but its polysaccharide viscosity has increase in various degree, and increasing and the raising of penbritin concentration along with passage number, the mutant strain that screening obtains is forward mutation, two kinds of polysaccharide yield and all obviously raisings of fermentation broth viscosity of the mutant strain of homopolysaccharide generation bacterium that all random screenings obtain.
Claims (3)
1. cultural method that improves bacterial polysaccharides output and viscosity, described method comprises:
(1) chooses the bacterium conduct bacterial strain to be cultivated that produces exocellular polysaccharide, measure penbritin to the minimal inhibitory concentration of this bacterial strain, be designated as MIC;
(2) will wait to cultivate inoculation to the appropriate liquid substratum, add the concentration penbritin of 1/3～2/3 concentration of the corresponding MIC of bacterial strain for this reason, cultivate 24h in 30 ℃, 200rpm, the gained nutrient solution is transferred and is cultivated under the same terms in the liquid nutrient medium that adds the same concentrations penbritin with the inoculum size of 10% volume ratio;
(3) operation of repeating step (2), corotation connects and cultivates 5 times under this penbritin concentration;
(4) improve penbritin concentration 5～20 μ g/ml on step (2) basis, according to step (2) method, corotation connects and cultivates 5 times under this penbritin concentration;
(5) improve penbritin concentration 5～20 μ g/ml on step (4) basis, according to step (2) method, corotation connects and cultivates 5 times under this penbritin concentration;
(6) operation of repeating step (5) improves constantly the cultivation of transferring of penbritin concentration, and corotation connects to be cultivated 100～200 times.
2. the method for claim 1, it is characterized in that described liquid nutrient medium is composed as follows: peptone 5g/L, Fructus Hordei Germinatus soak powder 3g/L, glucose 10g/L, yeast powder 3g/L, solvent are water, pH7.0.
3. the method for claim 1, the bacterium that it is characterized in that described product exocellular polysaccharide is xanthomonas campestris or Sphingol single-cell.
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| CN105385174A (en) * | 2015-10-27 | 2016-03-09 | 武汉理工大学 | Asphalt thickener and asphalt cement |
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| CN1351172A (en) * | 2000-10-26 | 2002-05-29 | 上海众伟生化有限公司 | Process for preparing microbial polyose jelly |
| CN101501213A (en) * | 2005-02-04 | 2009-08-05 | Cp凯尔科美国公司 | Targeted gene deletions for polysaccharide slime formers |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1351172A (en) * | 2000-10-26 | 2002-05-29 | 上海众伟生化有限公司 | Process for preparing microbial polyose jelly |
| CN101501213A (en) * | 2005-02-04 | 2009-08-05 | Cp凯尔科美国公司 | Targeted gene deletions for polysaccharide slime formers |
| US20110171721A1 (en) * | 2005-02-04 | 2011-07-14 | Cp Kelco U.S., Inc. | Targeted Gene Deletions for Polysaccharide Slime Formers |
| US20110281334A1 (en) * | 2005-02-04 | 2011-11-17 | C.P. Kelco U.S., Inc. | Targeted gene deletions for polysaccharide slime formers |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105385174A (en) * | 2015-10-27 | 2016-03-09 | 武汉理工大学 | Asphalt thickener and asphalt cement |
| CN105385174B (en) * | 2015-10-27 | 2017-10-24 | 武汉理工大学 | A kind of pitch thickener and bituminous cements |
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