CN102952834A - Method for producing microbial polysaccharide fermentation broth by using Paenibacillus mucilaginosus - Google Patents
Method for producing microbial polysaccharide fermentation broth by using Paenibacillus mucilaginosus Download PDFInfo
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Abstract
The present invention belongs to microbial polysaccharide fermentation, and particularly relates to a method for producing a microbial polysaccharide fermentation broth by using Paenibacillus mucilaginosus, wherein Paenibacillus mucilaginosus is inoculated in a culture medium containing a carbon source and a nitrogen source to carry out fermentation, and bacteria heat stimulation, addition of a surfactant during a fermentation process, and other manners are performed to optimize the production process. With the present invention, the problem of low yield of polysaccharides in the fermentation broth in the prior art is solved, and advantages of high polysaccharide yield in the fermentation broth, good bacterial growth, short fermentation period and the like are provided.
Description
Technical field
The invention belongs to the fermentation of microbial polysaccharide, refer to especially a kind of method of utilizing the colloid series bacillus to produce microbial polysaccharide fermentation liquid.
Background technology
The gel-shaped series bacillus is Soil Bacillus, and its bacterium colony is circular, and is colourless, protuberance, and the colloid thickness, transparent or semitransparent, neat in edge.The major function bacterial strain that is used as in the past few decades microbial fertilizer is exploited, and it has brought into play vital role in the agricultural microorganism formulation art.Along with deepening continuously that the colloid series bacillus is studied, people begin to note research and the application of its exocellular polysaccharide in recent years.But focus still concentrates on colloid bacillus cereus is produced the analysis of polysaccharide component and polysaccharide to the capacity of decomposition aspect of silicate minerals.The research that the new Application Areas of its polysaccharide and Optimization Technology is increased the polysaccharide yield aspect is also less.People mainly are switching inclined plane method activated inclined plane bacterial classifications to the traditional technology of microbial polysaccharide generation at present, optimize the conventional steps such as shake flask fermentation processing parameter, utilize the yield level of the polysaccharide in fermentation liquid of aforesaid method production to be 3-5g/L.
Summary of the invention
The object of the present invention is to provide the high colloid series bacillus that utilizes of microbial polysaccharide content in a kind of fermented liquid to produce the method for microbial polysaccharide fermentation liquid.
Overall technology design of the present invention is:
Utilize the colloid series bacillus to produce the method for microbial polysaccharide fermentation liquid, be with the colloid series bacillus (
Paenibacillus mucilaginosus) ACCC10013 is inoculated in the substratum that contains carbon source and nitrogenous source and ferments, and comprises following processing step:
A, bacterial classification thermal stimulus
Under aseptic condition, the bacterial classification spore in the test tube slant is added sterilized water, vibration washes spore and makes spore suspension and place sterile chamber, the sterile chamber that spore suspension is housed is placed boiling water, and homogeneous heating took out sterile chamber and rapidly cooling after 1-10 minute;
B, fermentation
The preparation of B1, substratum
Preparation substratum and sterilization are cooled to 30 ℃ with it, and substratum is comprised of the component of following mass percent:
Starch 5%-20%, sucrose 5%-10%, ammonium sulfate 0.5%-5%, dipotassium hydrogen phosphate 10%-20%, sal epsom 5%-10%, iron trichloride 0.01%-0.5%, calcium carbonate 0.1%-5%, yeast extract paste 0.1%-3%, surplus is sterilized water, pH=7.5;
B2, cultivation
In the substratum of step B preparation, fermentation period is 60 hours with the bacterial classification inoculation after processing in the steps A, and 30 ℃ of culture temperature, inoculum size are bacterial classification: the mass percent of substratum is 8% after the sterilization; Air quantity is adjusted to: (ventilating ratio is fermentating liquid volume: per minute passes into the volume of air to 0-4 hour ventilating ratio, and is lower same.) be 1:0.2-0.5VVM, 4-12 hour ventilating ratio is 1:0.5-0.7VVM, 12-20 hour ventilating ratio is 1:0.7-0.9VVM, 20-36 hour ventilating ratio is 1:1.1-1.5VVM, 28-36 hour ventilating ratio is 1:0.5-0.7VVM, 36-60 hour ventilating ratio is 1:0.3-0.5VVM, finishes to cultivate, and feed supplement stream adds tensio-active agent sodium laurylsulfonate 1-10ml after cultivating 36 hours.
Concrete technology contents among the present invention also has:
Sterile chamber can be selected including, but not limited to test tube or other containers of being convenient to heat, and for ease of the heating of sterile chamber, preferred technical scheme is that the sterile chamber in the steps A is selected test tube.
Homogeneous heating can adopt multiple existing mode, does not all break away from essence of the present invention, and wherein comparatively preferred technical scheme is, the homogeneous heating in the steps A adopts the mode of limit heating edge vibration to carry out.
Sterile chamber is taken out and cools off rapidly is to adopt cold water that sterile chamber is cooled off.
Charge amount is 38 liters of 50 liters of canister chargings among the step B.
The substantive distinguishing features that the present invention possesses and significant technical progress are:
1, with the bacterial classification violet staining after the thermal stimulus, smear, microscopy finds that weak bacterium and the gemma that some are weak have reduced, gemma is more neat, and is painted darker, and the performance of fermenting at 50 liters of canisters is that gemma is sprouted soon, the delayed growth phase shortens, reduced total fermentation time, and the thalli growth synchronism is conducive to well the technology controlling and process of back, such as the adding of tensio-active agent.
2, the colloid series bacillus produces a large amount of pod membranes when nutrition is poor, its 5-10 that can reach the thalline size doubly, the method that the present invention has taked wave band to cultivate during the fermentation, namely in the early stage of cultivating to the good growing environment of bacterial classification to improve its biomass, in the later stage of fermentation culture, strict control dissolved oxygen also adds tensio-active agent, creates oligotrophic environment and changes simultaneously membrane passage and make it at a large amount of accumulated polysaccharides of fermented liquid, to reach the purpose that improves polysaccharide yield.
3, after the fermentation ends, measure polysaccharide content.Detection method is the anthrone colorimetry, and its content reaches as high as 7.5g/L, can improve output of sugar 10-50% with the comparison of present report technique.
4, the fermented liquid of fermentation generation carries out preliminary extraction after separating with the sevag method and carries out the UV scanning detection, detects and finds that it does not contain nucleic acid and protein substantially.And tentatively record it and contain abundant hydroxyl and carboxyl, and the viscosity of this polysaccharide is high, similar with viscosity of xanthan gums, and it has good pseudo-plasticity and suspension, applies it to high-grade ceramic and makes, and finds that seldom this polysaccharide of amount can produce good effect, improve the smooth finish of ceramic surface, improve yield rate, the improving product class can produce good economic benefit.But the fermentation time with respect to this polysaccharide of xanthan gum is short, and the fermentation later stage is less to the demand of oxygen, has reduced because the aeration-agitation power consumption that the fermentation broth viscosity increase causes saves special equipment requirements.But this has started good field for the application that colloid bacillus cereus produces polysaccharide.In addition, since its high viscosity that has, the characteristics such as pseudo-plasticity and suspension, it can also be in petroleum prospecting, and also there is good application prospect the mineral floating aspect.
Embodiment
Do further and description below in conjunction with embodiments of the invention; but not as a limitation of the invention; protection scope of the present invention is as the criterion with the content of claim record, and any equivalence techniques means of making according to specification sheets are replaced, and all do not break away from protection scope of the present invention.
Embodiment 1
Utilize the colloid series bacillus to produce the method for microbial polysaccharide fermentation liquid, be with the colloid series bacillus (
Paenibacillus mucilaginosus) ACCC10013 is inoculated in the substratum that contains carbon source and nitrogenous source and ferments, and comprises following processing step:
A, bacterial classification thermal stimulus
Under aseptic condition, the bacterial classification spore in the test tube slant is added sterilized water, vibration washes spore and makes spore suspension and place sterile chamber, the sterile chamber that spore suspension is housed is placed boiling water, and homogeneous heating took out sterile chamber and rapidly cooling after 1 minute;
B, fermentation
The preparation of B1, substratum
Preparation substratum and sterilization are cooled to 30 ℃ with it, and substratum is comprised of the component of following mass percent:
Starch 5%, sucrose 5%, ammonium sulfate 0.5%, dipotassium hydrogen phosphate 10%, sal epsom 5%, iron trichloride 0.01%, calcium carbonate 0.1%, yeast extract paste 0.1%, surplus is sterilized water, pH=7.5;
B2, cultivation
In the substratum of step B preparation, fermentation period is 60 hours with the bacterial classification inoculation after processing in the steps A, and 30 ℃ of culture temperature, inoculum size are bacterial classification: the mass percent of substratum is 8% after the sterilization; Air quantity is adjusted to: 0-4 hour ventilating ratio is 1:0.2VVM, 4-12 hour ventilating ratio is 1:0.5VVM, 12-20 hour ventilating ratio is 1:0.7VVM, 20-36 hour ventilating ratio is 1:1.1VVM, 28-36 hour ventilating ratio is 1:0.5VVM, 36-60 hour ventilating ratio is 1:0.3VVM, finishes to cultivate, and feed supplement stream adds tensio-active agent sodium laurylsulfonate 1ml after cultivating 36 hours.
Sterile chamber in the steps A is selected test tube.
Homogeneous heating in the steps A adopts the mode of limit heating edge vibration to carry out.
Sterile chamber is taken out and cools off rapidly is to adopt cold water that sterile chamber is cooled off.
Charge amount is 38 liters of 50 liters of canister chargings among the step B.
After the fermentation ends, adopt the polysaccharide content in the anthrone colorimetric method for determining fermented liquid.Its content can reach 6.85g/L.
Embodiment 2
In the steps A sterile chamber homogeneous heating after 10 minutes, is taken out sterile chamber and rapidly cooling in the present embodiment;
B, fermentation
The preparation of B1, substratum
Preparation substratum and sterilization are cooled to 30 ℃ with it, and substratum is comprised of the component of following mass percent:
Starch 20%, sucrose 10%, ammonium sulfate 5%, dipotassium hydrogen phosphate 20%, sal epsom 10%, iron trichloride 0.5%, calcium carbonate 5%, yeast extract paste 3%, surplus is sterilized water, pH=7.5;
B2, cultivation
In the substratum of step B preparation, fermentation period is 60 hours with the bacterial classification inoculation after processing in the steps A, and 30 ℃ of culture temperature, inoculum size are bacterial classification: the mass percent of substratum is 8% after the sterilization; Air quantity is adjusted to: 0-4 hour ventilating ratio is 1:.5VVM, 4-12 hour ventilating ratio is 1:0.7VVM, 12-20 hour ventilating ratio is 1:0.9VVM, 20-36 hour ventilating ratio is 1:1.5VVM, 28-36 hour ventilating ratio is 1:0.7VVM, 36-60 hour ventilating ratio is 1:0.5VVM, finishes to cultivate, and feed supplement stream adds tensio-active agent sodium laurylsulfonate 10ml after cultivating 36 hours.
All the other contents are with embodiment 1.
After the fermentation ends, adopt the polysaccharide content in the anthrone colorimetric method for determining fermented liquid.Its content can reach 7.2g/L.
Embodiment 3
The sterile chamber that the step of the present embodiment will be equipped with spore suspension places the boiling water homogeneous heating, after 8 minutes sterile chamber is taken out and rapidly cooling;
B, fermentation
The preparation of B1, substratum
Preparation substratum and sterilization are cooled to 30 ℃ with it, and substratum is comprised of the component of following mass percent:
Starch 15%, sucrose 8%, ammonium sulfate 3%, dipotassium hydrogen phosphate 15%, sal epsom 8%, iron trichloride 0.3%, calcium carbonate 3%, yeast extract paste 1%, surplus is sterilized water, pH=7.5;
B2, cultivation
In the substratum of step B preparation, fermentation period is 60 hours with the bacterial classification inoculation after processing in the steps A, and 30 ℃ of culture temperature, inoculum size are bacterial classification: the mass percent of substratum is 8% after the sterilization; Air quantity is adjusted to: 0-4 hour ventilating ratio is 1:0.3VVM, 4-12 hour ventilating ratio is 1:0.6VVM, 12-20 hour ventilating ratio is 1:0.8VVM, 20-36 hour ventilating ratio is 1:1.3VVM, 28-36 hour ventilating ratio is 1:0.6VVM, 36-60 hour ventilating ratio is 1:0.4VVM, finishes to cultivate, and feed supplement stream adds tensio-active agent sodium laurylsulfonate 8ml after cultivating 36 hours.
All the other contents are with embodiment 1.
After the fermentation ends, adopt the polysaccharide content in the anthrone colorimetric method for determining fermented liquid.Its content can reach 7.5g/L.
Embodiment 4
The sterile chamber that the step of the present embodiment will be equipped with spore suspension places the boiling water homogeneous heating, after 6 minutes sterile chamber is taken out and rapidly cooling;
B, fermentation
The preparation of B1, substratum
Preparation substratum and sterilization are cooled to 30 ℃ with it, and substratum is comprised of the component of following mass percent:
Starch 8%, sucrose 6%, ammonium sulfate 1%, dipotassium hydrogen phosphate 13%, sal epsom 7%, iron trichloride 0.1%, calcium carbonate 1%, yeast extract paste 0.5%, surplus is sterilized water, pH=7.5;
B2, cultivation
In the substratum of step B preparation, fermentation period is 60 hours with the bacterial classification inoculation after processing in the steps A, and 30 ℃ of culture temperature, inoculum size are bacterial classification: the mass percent of substratum is 8% after the sterilization; Air quantity is adjusted to: 0-4 hour ventilating ratio is 1:0.3VVM, 4-12 hour ventilating ratio is 1:0.6VVM, 12-20 hour ventilating ratio is 1:0.8VVM, 20-36 hour ventilating ratio is 1:1.3VVM, 28-36 hour ventilating ratio is 1:0.6VVM, 36-60 hour ventilating ratio is 1:0.5VVM, finishes to cultivate, and feed supplement stream adds tensio-active agent sodium laurylsulfonate 3ml after cultivating 36 hours.
All the other contents are with embodiment 1.
After the fermentation ends, adopt the polysaccharide content in the anthrone colorimetric method for determining fermented liquid.Its content can reach 7.0g/L.
Embodiment 5
The sterile chamber that the step of the present embodiment will be equipped with spore suspension places the boiling water homogeneous heating, after 3 minutes sterile chamber is taken out and rapidly cooling;
B, fermentation
The preparation of B1, substratum
Preparation substratum and sterilization are cooled to 30 ℃ with it, and substratum is comprised of the component of following mass percent:
Starch 18%, sucrose 9%, ammonium sulfate 0.6%, dipotassium hydrogen phosphate 18%, sal epsom 9%, iron trichloride 0.07%, calcium carbonate 0.9%, yeast extract paste 0.8%, surplus is sterilized water, pH=7.5;
B2, cultivation
In the substratum of step B preparation, fermentation period is 60 hours with the bacterial classification inoculation after processing in the steps A, and 30 ℃ of culture temperature, inoculum size are bacterial classification: the mass percent of substratum is 8% after the sterilization; Air quantity is adjusted to: 0-4 hour ventilating ratio is 1:0.4VVM, 4-12 hour ventilating ratio is 1:0.6VVM, 12-20 hour ventilating ratio is 1:0.85VVM, 20-36 hour ventilating ratio is 1:1.45VVM, 28-36 hour ventilating ratio is 1:0.6VVM, 36-60 hour ventilating ratio is 1:0.35VVM, finishes to cultivate, and feed supplement stream adds tensio-active agent sodium laurylsulfonate 9ml after cultivating 36 hours.
All the other contents are with embodiment 1.
After the fermentation ends, adopt the polysaccharide content in the anthrone colorimetric method for determining fermented liquid.Its content can reach 6.95g/L.
Claims (5)
1. utilize the colloid series bacillus to produce the method for microbial polysaccharide fermentation liquid, be with the colloid series bacillus (
Paenibacillus mucilaginosus) ACCC10013 is inoculated in the substratum that contains carbon source and nitrogenous source and ferments, and it is characterized in that comprising following processing step:
A, bacterial classification thermal stimulus
Under aseptic condition, the bacterial classification spore in the test tube slant is added the 6ml sterilized water, vibration washes spore and makes spore suspension and place sterile chamber, the sterile chamber that spore suspension is housed is placed boiling water, and homogeneous heating took out sterile chamber and rapidly cooling after 1-10 minute;
B, fermentation
The preparation of B1, substratum
Preparation substratum and sterilization are cooled to 30 ℃ with it, and substratum is comprised of the component of following mass percent:
Starch 5%-20%, sucrose 5%-10%, ammonium sulfate 0.5%-5%, dipotassium hydrogen phosphate 10%-20%, sal epsom 5%-10%, iron trichloride 0.01%-0.5%, calcium carbonate 0.1%-5%, yeast extract paste 0.1%-3%, surplus is sterilized water, pH=7.5;
B2, cultivation
In the substratum of step B preparation, fermentation period is 60 hours with the bacterial classification inoculation after processing in the steps A, and 30 ℃ of culture temperature, inoculum size are bacterial classification: the mass percent of substratum is 8% after the sterilization; Air quantity is adjusted to: 0-4 hour ventilating ratio is 1:0.2-0.5VVM, 4-12 hour ventilating ratio is 1:0.5-0.7VVM, 12-20 hour ventilating ratio is 1:0.7-0.9VVM, 20-36 hour ventilating ratio is 1:1.1-1.5VVM, 28-36 hour ventilating ratio is 1:0.5-0.7VVM, 36-60 hour ventilating ratio is 1:0.3-0.5VVM, finishes to cultivate, and feed supplement stream adds tensio-active agent sodium laurylsulfonate 1-10ml after cultivating 36 hours.
2. the method for utilizing the colloid series bacillus to produce microbial polysaccharide fermentation liquid according to claim 1 is characterized in that the sterile chamber in the described steps A is selected test tube.
3. the method for utilizing the colloid series bacillus to produce microbial polysaccharide fermentation liquid according to claim 1 is characterized in that the homogeneous heating in the described steps A adopts the mode of limit heating edge vibration to carry out.
4. each described method of utilizing the colloid series bacillus to produce microbial polysaccharide fermentation liquid according to claim 1-3 is characterized in that described is to adopt cold water that sterile chamber is cooled off with sterile chamber taking-up and rapid cooling.
5. the method for utilizing the colloid series bacillus to produce microbial polysaccharide fermentation liquid according to claim 1 is characterized in that charge amount is 38 liters of 50 liters of canister chargings among the described step B.
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| CN103194410A (en) * | 2013-04-04 | 2013-07-10 | 河北省微生物研究所 | Paenibacillus mucilaginosus and method for producing compound microorganism bacterium agent by utilizing same |
| CN103642873A (en) * | 2013-12-05 | 2014-03-19 | 河北省微生物研究所 | Method for producing microbial flocculant by using Paenibacillus mucilaginosus |
| CN103740618A (en) * | 2013-12-31 | 2014-04-23 | 光明乳业股份有限公司 | Novel bacillus-like strain as well as culture method and application thereof |
| CN113151038A (en) * | 2021-01-13 | 2021-07-23 | 广东省农业科学院农业资源与环境研究所 | Strain for producing extracellular polysaccharide, method for producing extracellular polysaccharide by using strain and application |
| CN113477408A (en) * | 2021-07-21 | 2021-10-08 | 东北大学 | Application of curdlan serving as inhibitor in iron ore reverse flotation in mineral processing field and application method |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103194410A (en) * | 2013-04-04 | 2013-07-10 | 河北省微生物研究所 | Paenibacillus mucilaginosus and method for producing compound microorganism bacterium agent by utilizing same |
| CN103642873A (en) * | 2013-12-05 | 2014-03-19 | 河北省微生物研究所 | Method for producing microbial flocculant by using Paenibacillus mucilaginosus |
| CN103740618A (en) * | 2013-12-31 | 2014-04-23 | 光明乳业股份有限公司 | Novel bacillus-like strain as well as culture method and application thereof |
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| CN113151038A (en) * | 2021-01-13 | 2021-07-23 | 广东省农业科学院农业资源与环境研究所 | Strain for producing extracellular polysaccharide, method for producing extracellular polysaccharide by using strain and application |
| CN113477408A (en) * | 2021-07-21 | 2021-10-08 | 东北大学 | Application of curdlan serving as inhibitor in iron ore reverse flotation in mineral processing field and application method |
| CN113477408B (en) * | 2021-07-21 | 2022-08-02 | 东北大学 | Application of curdlan serving as inhibitor in iron ore reverse flotation in mineral processing field and application method |
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