CN100475971C - Preparing process of cold-adaptive deep sea microbe exopolysaccharide - Google Patents
Preparing process of cold-adaptive deep sea microbe exopolysaccharide Download PDFInfo
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Abstract
The present invention belongs to the field of marine biotechnology, and is preparation process of deep sea cold-adaptive microbe amylovorin. Separated and purified amylovorin is prepared through deep liquid fermentation with deep sea cold-adaptive bacterium strain Pseudomonas sp.SM9913 capable of resulting in high amylovorin yield. The structure of the amylovorin is analyzed and has introduced acetyl group to extend the molecule, expose the hydroxyl radical, increase the water solubility and raise the bioactivity. The purified product of the present invention may be used widely in medicine, food, environment protection, cosmetics and other fields.
Description
Technical field
The present invention relates to a kind of preparation method of new cold-adaptive deep sea microbe amylovorin, belong to the marine biotechnology field.
Background technology
60% ocean is that the degree of depth surpasses 2000 meters deep-sea, and the deep-sea is a special ecotope, permanent here low temperature (deep-sea hydrothermal port is a hot environment), high pressure, dark, high salt, oligotrophic.The multiple extreme microorganism of living in the deep-sea is such as having a liking for (anti-) hot bacterium, have a liking for (fitting) cold bacterium, having a liking for (anti-) and press bacterium, have a liking for (anti-) salt bacterium etc.The microorganism of deep-sea hydrothermal port area is the hot fields of studying at present.
Past 10 years, scientist has carried out a large amount of research to the structure and the function of the exocellular polysaccharide (Exopolysaccharides EPSs) of deep-sea hydrothermal port microorganism secretion, mainly is near the mesophilic bacteria excretory polysaccharide the hydrothermal solution mouth: alternately zygosaccharomyces, Pseudoalteromonas belong to and Vibrio.The structure of the exocellular polysaccharide of microorganism secretion is different in the exocellular polysaccharide of hydrothermal solution mouth microorganism secretion and other ecotopes; show glucuronic acid content height (up to 10～40%) and highly acetylated on; make that these polysaccharide viscosity are higher, metal ion, microbial cells self and protein grain are had stronger binding ability.The Microbial resources of deep-sea hydrothermal port are considered to secrete the valuable source of novel specific function polysaccharide.Because on the one hand, the special construction of the exocellular polysaccharide of deep-sea hydrothermal port microorganism secretion indicates that it has new potential biological function, will have new purposes in biological technical field; On the other hand, exocellular polysaccharide can be used as the type material of studying biomacromolecule stability protection under the extreme environment under the deep-sea extreme environment.Therefore, the Deep-Sea Microorganisms STUDY ON POLYSACHAROSE has caused various countries scientists' attention day by day, has become the focus of microbiology area research, simultaneously, also becomes various countries' competition focal point.China should strengthen the Deep-Sea Microorganisms STUDY ON POLYSACHAROSE, thereby can occupy a tiny space in this marine organisms high-tech sector.
Microbial polysaccharide is important biological technology products, has in fields such as medicine, biological technical field, food, environmental protection, makeup widely to use.The kind of microbial polysaccharide is a lot, its function of the structures shape of polysaccharide.The zone, deep-sea is owing to unique extreme ecosystem characterization such as its high pressure, high salt, low temperature, and therefore, Deep-Sea Microorganisms excretory exocellular polysaccharide should have new structural performance.Past is paid close attention to the structure and the effect in microorganism adaptation hydrothermal solution mouth extreme environment thereof of the exocellular polysaccharide that more is the deep-sea hydrothermal port microorganism secretion.But, deep-sea hydrothermal port in seabed, whole deep-sea shared area seldom, and, all be to have a liking for (anti-) hot bacterium or mesophilic bacteria mostly in the microorganism of this region growing.And overwhelming majority zone, seabed, deep-sea is the environment of a high pressure, low temperature, the low light level, oligotrophic marine stream, and these region growings the cold microorganism of a large amount of having a liking for (fitting).
Calendar year 2001, people's reported first such as Chen Xiulan bacterial strain screening and feature and the evaluation of deep sea cold-adaptive microbe bacterium strain Pseudomonas sp.SM9913, referring to Chen Xiulan etc., " low-temperature protease that deep sea cold-adaptive microbe bacterium strain SM9913 produces ", " ocean science " calendar year 2001, the 5th volume, the 1st phase, the 4-8 page or leaf.Chinese patent 01127405.0 discloses the method for utilizing this bacterial strain to produce properly cooled proteinase with special flavor.But up to the present, the research to the structure and the function of cold-adaptive deep sea microbe amylovorin does not appear in the newspapers as yet, and to the research of this problem, has more general significance for the Deep-Sea Microorganisms utilization, is worth deeply inquiring into.
The invention provides a kind of preparation method of cold-adaptive deep sea microbe amylovorin.
It is bacterial strain that the present invention at first provides with the high deep sea cold-adaptive microbe bacterium strain Pseudomonas sp.SM9913 of exocellular polysaccharide productive rate, the technology for preparing exocellular polysaccharide by liquid submerged fermentation, obtain the exocellular polysaccharide of separation and purification, resolve its structure, for its application at biological technical field lays the foundation.
Technical scheme of the present invention is as follows:
A kind of preparation method of cold-adaptive deep sea microbe amylovorin comprises the steps:
(1) seed preparation
Substratum: 2～3 parts of soybean cake powder, 2～3 parts of corns, 1～1.5 part in wheat bran, Na
2HPO
40.4～0.5 part, KH
2PO
40.03 100 parts in～0.04 part and water, be weight part, sterilized 30-50 minute for substratum 100-120 ℃, after the cooling, with deep sea cold-adaptive microbe bacterium strain Pseudomonas sp.SM9913 be bacterial strain (referring to Chen Xiulan etc., " low-temperature protease that deep sea cold-adaptive microbe bacterium strain SM9913 produces ", on " ocean science " January calendar year 2001 19, bacterial strain possessor Shandong University), connect the bacteria suspension of eggplant bottle bacterial classification, inoculum size 5-7% mass percent.Ventilate, stir, cultivate.Get seed liquor, stand-by.
Preferably, add soya-bean oil in the above-mentioned substratum and make defoamer for 300～350 milliliters.
Preferably, the preparation of above-mentioned seed is 70 liters of sample-loading amounts in 150 liters of fermentor tanks, under 10～12 ℃, ventilates 1: 0.5～0.6, stirs 320～330 rev/mins, cultivates 36～48 hours.About 5 grams per liters of exopolysaccharides.
(2) liquid submerged fermentation prepares the exocellular polysaccharide fermented liquid
Substratum: 3～3.5 parts of soybean cake powder, 2.5～3 parts of Semen Maydis powder, 2.5～3 parts of Rhizoma Dioscoreae powder, 2.0～2.5 parts in wheat bran, Na
2HPO
40.4～0.5 part, KH
2PO
40.03 100 parts in～0.04 part and water are weight part.1000 liters of fermentor tanks, adorn 450 liters of substratum, sterilization back pH6.2～6.3, the seed 5～7% of inoculation step (1) preparation stirs 200～220 rev/mins, control ventilation 0-12 hour 1: 0.5～0.6,12-20 hour 1: 0.6～0.7,20-28 hour 1: 0.8～0.9,10～12 ℃ of control culture temperature were fermented 48～72 hours.Every liter is produced the polysaccharide amount and can reach 5 grams.
Preferably, add soya-bean oil in the above-mentioned substratum and make defoamer for 1～1.2 kilogram.
(3) aftertreatment of exocellular polysaccharide fermented liquid
Above-mentioned exocellular polysaccharide fermented liquid is centrifugal through 10000rpm, and centrifuged supernatant precipitates with dehydrated alcohol then, and supernatant liquor volume and dehydrated alcohol volume ratio are 1: 3, and after albumen, decolouring, centrifugal drying obtains the polysaccharide raw product.Polysaccharide crude is heavy molten in 60～70 ℃ of hot water, is concentrated into 20～30% of original volume, separates with ion-exchange chromatography through gel permeation chromatography again, and vacuum lyophilization obtains the pure product of polysaccharide.
At last, through quality inspection, be distributed into the exocellular polysaccharide product.
The making of the eggplant bottle slant strains described in the above-mentioned steps (1) can be by prior art.The invention provides following concrete operations step:
With 0.2～0.4 portion of extractum carnis, 0.8～1.2 part of peptone, 1.5～2 parts of agar, 0.4～0.6 part of NaCl is substratum, 95～100 parts in water, pH is 7.0～7.2, sterilization, on the test tube slant, line connects deep sea cold-adaptive microbe bacterium strain Pseudomonassp.SM9913 bacterial classification, cultivates 24～26 hours, and preserves in 4 ℃ for 12～15 ℃.
Above-mentioned bacterial classification is moved into eggplant bottle inclined-plane (substratum is identical with the preservation inclined-plane), cultivated 24～26 hours for 12～15 ℃, make bacteria suspension and use.
Preferred in above-mentioned steps (3) gel permeation chromatography and the ion exchange chromatography:
Gel permeation chromatography post: 100cm * 1.2cm, gel type: Sephadex G-100.
Ion exchange column: 25cm * 1.6cm, gel type: DEAE-Sepharose Fast Flow.
Below in conjunction with the structure elucidation of exocellular polysaccharide, the present invention will be further described.
The prepared pure product of polysaccharide of the inventive method through the primary structure composition repeating unit that 1D, 2D-NMR, MS, methylation analysis obtain this polysaccharide are:
{→6)-[3，6-O-acetyl]-α-D-Glcp-(1→6)-[3-O-acetyl]-α-D-Glcp-(1→6)-[3-O-acetyl]-α-D-Glcp-(1→6)-[3-O-acetyl]-α-D-Glcp-(1→}
n
Sugar unit wherein: ethanoyl: ethanol based=4: 5: 1; highly acetylated α-(1 → 6) constitutes the core texture (61.8%) of polysaccharide main chain, and the end of polysaccharide chain also exists a spot of Ara (11.0%), Xyl (3.9%), Gal (3.1%), (1 → 4) Glc (5%) and (3 → 6) Glc (4%).From then in the exocellular polysaccharide structure; the highly acetylated as can be seen directional property and the horizontal order that can change polysaccharide molecule; thereby change the physical properties of polysaccharide; the introducing of ethanoyl changes the stretching, extension of molecule; finally cause the exposure of polysaccharide oh group; increase its solvability in water, improve its bioactive effect thereby reach.
The pure product of prepared polysaccharide of the present invention are biological technology products, have in fields such as medicine, biological technical field, food, environmental protection, makeup widely to use.
Technical characterstic of the present invention is:
1, the ocean is as a special extreme environment, and marine microorganism excretory EPS should have special structure and function, has caused various countries scientist's concern day by day.In recent years, the middle temperature of deep-sea hydrothermal port and structure and the function of heat resistant microbe and polar region psychrophiles excretory EPS are carried out many researchs, found the EPS of multiple new 26S Proteasome Structure and Function.But, do not appear in the newspapers so far about the structure of deep-sea psychrophiles excretory EPS and the research of function.Because the environment more than 60% all is that the degree of depth surpasses 2000 meters deep-sea in the world, wherein the overwhelming majority all is a low temperature environment.
2, be that strain fermentation is produced exocellular polysaccharide with deep sea cold-adaptive microbe bacterium strain Pseudomonas sp.SM9913 first, set up the optimization fermentation manufacturing technique of the exocellular polysaccharide EPS of this bacterial strain synthesis secretion.
3, the technological method of high efficiency separation purifying deep sea cold-adaptive microbe bacterium strain Pseudomonas sp.SM9913 excretory exocellular polysaccharide is provided first, utilize technology such as nucleus magnetic resonance, infrared spectra, mass spectrum, analyze its physics-chem characteristic, resolve its structure, the primary structure of polysaccharide is formed as previously mentioned, illustrates that this is a kind of Deep-Sea Microorganisms exocellular polysaccharide of new structure.
Embodiment
The present invention will be further described below in conjunction with embodiment, but be not limited thereto.
Embodiment 1: the preparation of the bacteria suspension of eggplant bottle bacterial classification
With 0.3 portion of extractum carnis, 1.0 parts of peptones, 1.7 parts of agar, 0.5 part of NaCl is substratum, 96 parts in water, pH is 7.0～7.2, and sterilization is on the test tube slant, line connects deep sea cold-adaptive microbe bacterium strain Pseudomonas sp.SM9913 bacterial classification (referring to Chen Xiulan etc., " low-temperature protease that deep sea cold-adaptive microbe bacterium strain SM9913 produces ", 19 days " ocean science " January calendar year 2001, bacterial strain possessor Shandong University), cultivated 25 hours, and preserved for 12～15 ℃ in 4 ℃.This bacterial classification is moved into eggplant bottle inclined-plane (substratum is identical with the preservation inclined-plane), cultivated 25 hours for 12～15 ℃, make bacteria suspension and use.
Embodiment 2: the preparation of cold-adaptive deep sea microbe amylovorin, and step is as follows:
(1) seed preparation
Substratum: 2 parts of soybean cake powder, 3 parts of corns, 1.5 parts in wheat bran, Na
2HPO
40.5 part, KH
2PO
40.04 100 parts in part and water are weight part, add soya-bean oil in the substratum and make defoamer for 300 milliliters.120 ℃ of sterilizations of substratum 50 minutes, after the cooling, sample-loading amount is 70 liters in 150 liters of fermentor tanks, connects the bacteria suspension of the eggplant bottle bacterial classification of embodiment 1 preparation, inoculum size 5%.Under 10～12 ℃, ventilate 1: 0.5～0.6, stir 320～330 rev/mins, cultivated 40 hours.
(2) liquid submerged fermentation prepares the exocellular polysaccharide fermented liquid
Substratum: 3.5 parts of soybean cake powder, 2.5 parts of Semen Maydis powder, 3 parts of Rhizoma Dioscoreae powder, 2.5 parts in wheat bran, Na
2HPO
40.4 part, KH
2PO
40.03 100 parts in part and water are weight part.Add soya-bean oil in the substratum and make defoamer for 1 kilogram.1000 liters of fermentor tanks, adorn 450 liters of substratum, sterilization back pH6.2～6.3, the seed of inoculation step (1) preparation, inoculum size 5%, stir 220 rev/mins, control ventilation 0-12 hour 1: 0.5～0.6,12-20 hour 1: 0.6～0.7,20-28 hour 1: 0.8～0.9,10～12 ℃ of control culture temperature were fermented 56 hours.Every liter is produced the polysaccharide amount and can reach 5 grams.
(3) aftertreatment of exocellular polysaccharide fermented liquid
Above-mentioned exocellular polysaccharide fermented liquid is centrifugal through 10000rpm, and then with dehydrated alcohol precipitation (centrifuged supernatant volume and dehydrated alcohol volume ratio are 1: 3), after albumen, decolouring, centrifugal drying obtains the raw product of polysaccharide.Polysaccharide crude is heavy molten in 60～70 ℃ of hot water, is concentrated into 22% of original volume, separates with ion-exchange chromatography through gel permeation chromatography again, and vacuum lyophilization obtains the pure product (solid) of polysaccharide.Preferred in gel permeation chromatography and the ion exchange chromatography: gel permeation chromatography post: 100cm * 1.2cm, gel type: Sephadex G-100; Ion exchange column: 25cm * 1.6cm, gel type: DEAE-Sepharose Fast Flow.
The pure product of prepared polysaccharide through the primary structure composition repeating unit that 1D, 2D-NMR, MS, methylation analysis obtain this polysaccharide are:
{→6)-[3，6-O-acetyl]-α-D-Glcp-(1→6)-[3-O-acetyl]-α-D-Glcp-(1→6)-[3-O-acetyl]-α-D-Glcp-(1→6)-[3-O-acetyl]-α-D-Glcp-(1→)
n
Sugar unit wherein: ethanoyl: ethanol based=4: 5: 1; highly acetylated α-(1 → 6) constitutes the core texture (61.8%) of polysaccharide main chain, and the end of polysaccharide chain also exists a spot of Ara (11.0%), Xyl (3.9%), Gal (3.1%), (1 → 4) Glc (5%) and (3 → 6) Glc (4%).
Embodiment 3:
The preparation method of cold-adaptive deep sea microbe amylovorin comprises the steps:
(1) seed preparation
Substratum: 2 parts of soybean cake powder, 3 parts of corns, 1.5 parts in wheat bran, Na
2HPO
40.5 part, KH
2PO
40.04 100 parts in part and water are weight part, add soya-bean oil in the substratum and make defoamer for 300 milliliters.120 ℃ of sterilizations of substratum 50 minutes, after the cooling, the bacteria suspension (can be the product of embodiment 1) of inoculation SM9913 bacterial classification eggplant bottle bacterial classification, inoculum size 5% mass percent.The preparation of above-mentioned seed is 70 liters of sample-loading amounts in 150 liters of fermentor tanks, under 10～12 ℃, ventilates 1: 0.5～0.6, stirs 320～330 rev/mins, cultivates 48 hours.Exopolysaccharides 5 grams per liters.
(2) liquid submerged fermentation prepares the exocellular polysaccharide fermented liquid
Substratum: 3.5 parts of soybean cake powder, 2.5 parts of Semen Maydis powder, 3 parts of Rhizoma Dioscoreae powder, 2.5 parts in wheat bran, Na
2HPO
40.4 part, KH
2PO
40.03 100 parts in part and water are weight part, add soya-bean oil in the substratum and make defoamer for 1.2 kilograms.1000 liters of fermentor tanks, adorn 450 liters of substratum, sterilization back pH6.2～6.3, the seed of inoculation step (1) preparation, inoculum size 6%, stir 200～220 rev/mins, control ventilation 0-12 hour 1: 0.5～0.6,12-20 hour 1: 0.6～0.7,20-28 hour 1: 0.8～0.9,10～12 ℃ of control culture temperature were fermented 72 hours.Every liter is produced the polysaccharide amount and can reach 5 grams.
(3) aftertreatment of exocellular polysaccharide fermented liquid
Above-mentioned exocellular polysaccharide fermented liquid is centrifugal through 10000rpm, and then with dehydrated alcohol precipitation (centrifuged supernatant volume and dehydrated alcohol volume ratio are 1: 3), after albumen, decolouring, centrifugal drying obtains the raw product of polysaccharide.Polysaccharide crude is heavy molten in 65 ℃ of hot water, is concentrated into 30% of original volume, separates with ion-exchange chromatography through gel permeation chromatography again, and vacuum lyophilization obtains the pure product of polysaccharide.Preferred in gel permeation chromatography and the ion exchange chromatography: gel permeation chromatography post: 100cm * 1.2cm, gel type: Sephadex G-100; Ion exchange column: 25cm * 1.6cm, gel type: DEAE-Sepharose Fast Flow.
Polysaccharide structures is formed with embodiment 2.
Claims (7)
1, a kind of preparation method of cold-adaptive deep sea microbe amylovorin comprises the steps:
(1) seed preparation
Substratum: 2～3 parts of soybean cake powder, 2～3 parts of corns, 1～1.5 part in wheat bran, Na
2HPO
40.4～0.5 part, KH
2PO
40.03 100 parts in～0.04 part and water are weight part, substratum 100-120 ℃ of sterilization 30-50 minute after the cooling, is bacterial strain with deep sea cold-adaptive microbe bacterium strain Pseudomonas sp.SM9913, connects the bacteria suspension of eggplant bottle bacterial classification, inoculum size 5-7% mass percent; Ventilate, stir, cultivate; Get seed liquor, stand-by;
(2) liquid submerged fermentation prepares the exocellular polysaccharide fermented liquid
Substratum: 3～3.5 parts of soybean cake powder, 2.5～3 parts of Semen Maydis powder, 2.5～3 parts of Rhizoma Dioscoreae powder, 2.0～2.5 parts in wheat bran, Na
2HPO
40.4～0.5 part, KH
2PO
40.03 100 parts in～0.04 part and water are weight part; 1000 liters of fermentor tanks, adorn 450 liters of substratum, sterilization back pH6.2～6.3, the seed 5～7% of inoculation step (1) preparation stirs 200～220 rev/mins, control ventilation 0-12 hour 1: 0.5～0.6,12-20 hour 1: 0.6～0.7,20-28 hour 1: 0.8～0.9,10～12 ℃ of control culture temperature were fermented 48～72 hours;
(3) aftertreatment of exocellular polysaccharide fermented liquid
Above-mentioned exocellular polysaccharide fermented liquid is centrifugal through 10000rpm, and centrifuged supernatant precipitates with dehydrated alcohol then, and supernatant liquor volume and dehydrated alcohol volume ratio are 1: 3, and after albumen, decolouring, centrifugal drying obtains the polysaccharide raw product.Polysaccharide crude is heavy molten in 60～70 ℃ of hot water, is concentrated into 20～30% of original volume, separates with ion-exchange chromatography through gel permeation chromatography again, and vacuum lyophilization obtains the pure product of polysaccharide.
2, the preparation method of cold-adaptive deep sea microbe amylovorin as claimed in claim 1 is characterized in that, adds soya-bean oil in the substratum of step (1) and makes defoamer for 300～350 milliliters.
3, the preparation method of cold-adaptive deep sea microbe amylovorin as claimed in claim 1 is characterized in that, the preparation of the seed of step (1) is 70 liters of sample-loading amounts in 150 liters of fermentor tanks, under 10～12 ℃, ventilate 1: 0.5～0.6, stir 320～330 rev/mins, cultivated 36～48 hours.
4, the preparation method of cold-adaptive deep sea microbe amylovorin as claimed in claim 1 is characterized in that, the making concrete operations step of the eggplant bottle slant strains described in the step (1) is as follows:
With 0.2～0.4 portion of extractum carnis, 0.8～1.2 part of peptone, 1.5～2 parts of agar, 0.4～0.6 part of NaCl is substratum, 95～100 parts in water, pH is 7.0～7.2, sterilization, on the test tube slant, line connects deep sea cold-adaptive microbe bacterium strain Pseudomonassp.SM9913 bacterial classification, cultivates 24～26 hours, and preserves in 4 ℃ for 12～15 ℃; Above-mentioned bacterial classification is moved into eggplant bottle inclined-plane, and substratum is identical with the preservation inclined-plane, cultivates 24～26 hours for 12～15 ℃, makes bacteria suspension and uses.
5, the preparation method of cold-adaptive deep sea microbe amylovorin as claimed in claim 1 is characterized in that, adds soya-bean oil in step (2) substratum and makes defoamer for 1～1.2 kilogram.
6, the preparation method of cold-adaptive deep sea microbe amylovorin as claimed in claim 1 is characterized in that, the gel permeation chromatography of step (3) selects for use the gel permeation chromatography post to be: 100cm * 1.2cm, Sephadex G-100.
7, the preparation method of cold-adaptive deep sea microbe amylovorin as claimed in claim 1 is characterized in that, it is 25cm * 1.6cm that step (3) ion exchange chromatography is selected ion exchange column for use, DEAE-Sepharose Fast Flow.
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| CN102321689A (en) * | 2011-08-18 | 2012-01-18 | 山东福洋生物科技有限公司 | Semicontinuous fermentation technology of gluconate |
| CN102732583A (en) * | 2012-07-19 | 2012-10-17 | 山东大学 | Preparation method and application of deep-sea extracellular polymeric substances |
| CN103014052B (en) * | 2012-12-06 | 2014-06-18 | 山东大学 | Autonomously replicating expression vector pEV, and preparation method and application thereof |
| CN105647990B (en) * | 2016-03-14 | 2019-07-02 | 山东省食品发酵工业研究设计院 | A kind of Microbial exopolysaccharides and preparation method thereof |
| CN105858599B (en) * | 2016-04-18 | 2018-06-26 | 山东大学 | Seawater Psychrobacter synthesizes and carries out Bio-Nano-Materials of self assembly and preparation method and application |
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Non-Patent Citations (2)
| Title |
|---|
| 深海适冷菌Pseudoalteromonas sp. SM9913分泌的胞外多糖的分离纯化、结构鉴定及在适冷菌适应深海环境中的生态学功能研究. 秦国奎等.中国资源生物技术与糖工程学术研讨会论文集. 2005 |
| 深海适冷菌Pseudoalteromonas sp. SM9913分泌的胞外多糖的分离纯化、结构鉴定及在适冷菌适应深海环境中的生态学功能研究. 秦国奎等.中国资源生物技术与糖工程学术研讨会论文集. 2005 * |
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