CN103305566B - 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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- CN103305566B CN103305566B CN201310237997.8A CN201310237997A CN103305566B CN 103305566 B CN103305566 B CN 103305566B CN 201310237997 A CN201310237997 A CN 201310237997A CN 103305566 B CN103305566 B CN 103305566B
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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 to be secreted by bacterial cell, by multiple monosaccharide units or derivative through condensation, dehydration and the polymer coupling together.Bacterial polysaccharides is mainly present in occurring in nature with three kinds of forms: stick on cell surface, be secreted in substratum or 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, the monose that the latter is contained two or more.Bacterial polysaccharides is that fermentation using bacteria produces, and therefore, compared with animals and plants polysaccharide, its production is subject to the impact of the factors such as the 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 unique structure and good physicochemical property simultaneously, and therefore, in medicine, food, chemical industry, oil production, environment protection and other field have a wide range of applications.
Among all various extracellular polysaccharide of bacterias, gelling gum and xanthan gum are two kinds of the most successful polysaccharide of business development.Xanthan gum is the exocellular polysaccharide being produced by xanthomonas campestris (Xanthomnas campestris) fermentation.The glucose main chain that its structure is connected by β-(1 → 4) key and the side chain of trisaccharide unit form; Side chain is made up of seminose, glucuronic acid and seminose.The seminose that connects main chain is acetylation, and the hydroxyl of end seminose and the carbonyl of pyruvic acid form acetal.Gelling gum is the exocellular polysaccharide being produced through aerobic fermentation by Sphingol single-cell (Sphingomonas elodea).The linear anionic polysaccharide being formed by 3 kinds of monose repeated polymerization; that is: 2 molecule D-Glucoses; the L-rhamnosyl of 1 molecule D-Glucose aldehydic 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 foodstuff additive use by U.S. FDA and other multiple countries.
But because the output that current most polysaccharide produce bacterium is relatively low, production cost is higher, and its viscosity is lower, has affected to a certain extent its using value.Current study hotspot is to increase output by the whole bag of tricks to improve using value.Many investigators, by the transformation to polysaccharide biosynthetic pathway genes involved, have improved polysaccharide yield.But because gelling gum and xanthan gum etc. are as foodstuff additive, be genetic engineering modified bacterial strain if it produces bacterial strain, can cause the worry of human consumer to its security.And the genes involved of the biosynthetic pathway of some polysaccharide is not illustrated, cannot transform it by genetic engineering means.Also have research physics or chemical mutagen to process producing bacterial strain, but because these type of mutagenic compound are larger to cell injury, the positive variation rate that therefore obtains high yield high viscosity bacterial strain is lower, screening efficiency is low.Therefore, in the urgent need to a kind of high efficiency method of the non-genomic engineering with ubiquity, transform producing bacterial strain, screening can be produced has higher output yield and more full-bodied polysaccharide production bacterial strain.
Many cells microbiotic as the effect of penbritin under, can produce active oxygen, thus damage dna, produce sudden change.Therefore, penbritin also can be considered to a kind of relatively mild mutagenic compound.In addition, although various bacterial polysaccharides has various structure and monose composition, except a few polysaccharide, the first step in the biosynthetic pathway of other most of extracellular polysaccharide of bacterias and bacteria cell wall peptidoglycan there is similarity.The first step of transposon mutagenesis is that the nucleosides sugar (NDP-sugars) of activation is connected with the fat carrier on cytolemma, and then other nucleosides sugar of addition progressively, completes the assembling of repeating unit.This biosynthetic process is synthetic similar to bacteria cell wall moiety peptidoglycan.And the building-up process of peptidoglycan can be suppressed by microbiotic penbritin.In order to breed under the inhibition of penbritin, cell need synthesize the nucleosides sugar of more fat carrier and activation.Therefore penbritin there is the cell of resistance, may there is the nucleosides sugar synthesis capability of stronger fat carrier and activation, therefore under the condition existing at antibiotic-free, these how 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, is also a kind of synthetic more the more stress factors of sugar of cell that promotes simultaneously.
(3) summary of the invention
The object of the invention is to utilize penbritin can be simultaneously as the characteristic of mutagenic compound and the synthetic stress factors of bacteria cell wall peptidoglycan, a kind of cultural method that improves different bacterium polysaccharide yield and viscosity is provided, thereby continuous passage cultivate in bacterial polysaccharides produce the content that bacterium progressively improves penbritin and improve the stimulation to bacterial cell, further can be used for screening the full-bodied bacterial polysaccharides generation of high yield bacterium.
The technical solution used in the present invention is:
Improve a cultural method for bacterial polysaccharides output and viscosity, described method comprises:
(1) choose the bacterium that produces exocellular polysaccharide as bacterial strain to be cultivated, measure the minimal inhibitory concentration of penbritin to this bacterial strain, be designated as MIC;
(2) by inoculation to be cultivated in suitable liquid nutrient medium, add the concentration penbritin of 1/3～2/3 concentration of the corresponding MIC of bacterial strain for this reason, cultivate 24h in 30 DEG C, 200rpm, gained nutrient solution, with the inoculum size of 10% volume ratio, is transferred and under the same terms, is cultivated in the liquid nutrient medium that adds same concentrations penbritin; Described liquid nutrient medium is that routine is applicable to go down to posterity and cultivates the liquid nutrient medium that produces extracellular polysaccharide strains;
(3) operation of repeating step (2), under this penbritin concentration, corotation connects and cultivates 5 times;
(4) on step (2) basis, improve penbritin concentration 5～20 μ g/ml, according to step (2) method, under this penbritin concentration, corotation connects and cultivates 5 times;
(5) on step (4) basis, improve penbritin concentration 5～20 μ g/ml, according to step (2) method, under this penbritin concentration, corotation connects and cultivates 5 times;
(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 the bacterial strain screening, can, in every switching 30～50 end of taking turns and transfer, get nutrient solution, gradient dilution is in containing the YM flat board of same concentration penbritin (adding 15g/L agar in YM liquid nutrient medium) again, 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 soaks powder 3g/L, glucose 10g/L, yeast powder 3g/L, solvent is 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 the dual nature of gentle mutagenic compound and stress factors, improve various bacteria exopolysaccharides and viscosity.Penbritin of the present invention is coerced continuous passage method, process the bacterium of various product exocellular polysaccharides, especially in the situation that not knowing its biosynthetic pathway genes involved, improve polysaccharide yield and viscosity, and can avoid the worry of the harm that human consumer may bring genetic engineering means, be a kind of method that general extracellular polysaccharide of bacteria content and viscosity improve.Meanwhile, in the different treatment stage, the different strains that a series of produced polysaccharide viscosity increases gradually can be obtained, different application requiring can be adapted to.
(4) embodiment
Produces bacterium xanthomonas campestris and gelling gum taking xanthan gum below and produce bacterium Sphingol single-cell the present invention is described further in conjunction with specific embodiments as example, 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 purchased from ATCC) and Sphingol single-cell Sphingomonas elodea ATCC 31461(purchased from ATCC) be inoculated in YM liquid nutrient medium, 30 DEG C, 200rpm is cultured to logarithmic phase;
2, nutrient solution is switched on 96 orifice plates containing different concns penbritin (10～200 μ g/ml) with 1:1000 volume ratio, cultivates 24h for 30 DEG C;
3, measure the 0h cultivating and the optical density value OD that cultivates the 24h finishing by microplate reader respectively
595;
4, taking in aperture completely the lowest concentration of drug of bacteria growing inhibiting as 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 mutagenesis continuous passage and cultivated
1, xanthomonas campestris ATCC 13951 and Yi Le Sphingol single-cell ATCC 31461 are inoculated in YM liquid nutrient medium, and 30 DEG C, 200rpm cultivates 24h; YM liquid nutrient medium composition: peptone 5g/L, Fructus Hordei Germinatus soaks powder 3g/L, glucose 10g/L, yeast powder 3g/L, solvent is water, pH7.0;
2, respectively above-mentioned culture is transferred respectively in containing the YM liquid nutrient medium of 50 μ g/ml penbritins with 10% inoculum size, 30 DEG C, 200rpm cultivates 24h;
3, continue to transfer respectively in containing the YM liquid nutrient medium of 50 μ g/ml penbritins with 10% inoculum size, 30 DEG C, 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 in the YM liquid nutrient medium containing the penbritin of 50 μ g/ml after subculture 5 times, and the content of penbritin improves 10 μ g/ml, to total concn be 60 μ g/ml, 30 DEG C, 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 maximum tolerance concentration.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 mutagenesis continuous passage culturing process
1, draw respectively go down to posterity 55 times (penbritin concentration is 150 μ g/ml), 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 containing in the YM solid plate of corresponding penbritin concentration, cultivates 72h for 30 DEG C;
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 to containing in the inclined-plane of YM solid medium (adding again 15g/L agar in YM liquid nutrient medium), cultivate 72h for 30 DEG C, stand-by.
Embodiment 4: 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 YM liquid nutrient medium, cultivate 72h for 30 DEG C;
2, be forwarded in fermention medium separately with 10% inoculum size, cultivate 48h for 30 DEG C, 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 is 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 is water, pH7.0; 3, fermented sample is measured respectively polysaccharide yield and fermentation broth viscosity, and its measurement result sees the following form:
Table 1: xanthomonas campestris and Sphingol single-cell wild type strain and penbritin are coerced mutant strain leavening property comparison after mutagenesis
a
astrain number is as SE150G55-1 and XC150G55-1 etc., wherein SE represents Yi Le Sphingol single-cell mutant strain, XC represents xanthomonas campestris mutant strain, after SE 150,250,350 represent that respectively in the process that goes down to posterity, final penbritin concentration is respectively 150,250,350 μ g/ml, G55, G105 and G155 represent that respectively the number of times going down to posterity is 55,105 and 155 times ,-1～-20 represent be the difference list 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, although now the random raising that separates the polysaccharide yield that obtains mutant strain (gelling gum produces bacterium and xanthan gum produces bacterium) is not very obvious, 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 that homopolysaccharide does not produce the mutant strain of bacterium that all random screenings obtain.
Claims (2)
1. improve a cultural method for bacterial polysaccharides output and viscosity, described method comprises:
(1) choose the bacterium that produces exocellular polysaccharide as bacterial strain to be cultivated, measure the minimal inhibitory concentration of penbritin to this bacterial strain, be designated as MIC, the bacterium of described product exocellular polysaccharide is xanthomonas campestris Xanthomonas campestrisATCC 13951 or Sphingol single-cell Sphingomonas elodea ATCC 31461;
(2) by inoculation to be cultivated in suitable liquid nutrient medium, adding concentration is the penbritin of 50 μ g/ml, cultivate 24h in 30 DEG C, 200rpm, gained nutrient solution, with the inoculum size of 10% volume ratio, is transferred and under the same terms, is cultivated in the liquid nutrient medium that adds same concentrations penbritin;
(3) operation of repeating step (2), under this penbritin concentration, corotation connects and cultivates 5 times;
(4) on step (2) basis, improve penbritin concentration 10 μ g/ml, according to step (2) method, under this penbritin concentration, corotation connects and cultivates 5 times;
(5) on step (4) basis, improve penbritin concentration 10 μ g/ml, according to step (2) method, under this penbritin concentration, corotation connects and cultivates 5 times;
(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, is characterized in that described liquid nutrient medium is composed as follows: peptone 5g/L, and Fructus Hordei Germinatus soaks powder 3g/L, glucose 10g/L, yeast powder 3g/L, solvent is water, pH 7.0.
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| 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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| 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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