CN100475971C - A kind of preparation method of exopolysaccharide of deep-sea cold suitable microorganism - Google Patents

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

A kind of preparation method of exopolysaccharide of deep-sea cold suitable microorganism

technical field

The invention relates to a new method for preparing extracellular polysaccharides from deep-sea cold-suitable microorganisms, and belongs to the technical field of marine biology.

Background technique

60% of the ocean is the deep sea with a depth of more than 2000 meters. The deep sea is a special ecological environment, where there is permanent low temperature (the deep sea hydrothermal vent is a high temperature environment), high pressure, darkness, high salinity, and oligotrophic. A variety of extremophiles live in the deep sea, such as thermophilic (resistant) bacteria, psychrophilic (suitable) bacteria, barophilic (resistant) bacteria, halophilic (resistant) bacteria, etc. Microorganisms in deep-sea hydrothermal vent areas are currently a hot research area.

In the past ten years, scientists have conducted a lot of research on the structure and function of exopolysaccharides (Exopolysaccharides EPSs) secreted by microorganisms in deep-sea hydrothermal vents, mainly polysaccharides secreted by mesophilic bacteria near hydrothermal vents: Alternomonas, Pseudomonas Alteromonas and Vibrio. The structure of exopolysaccharides secreted by microorganisms in hydrothermal vents is different from those secreted by microorganisms in other ecological environments, which is manifested in high uronic acid content (up to 10-40%) and high acetylation, making these polysaccharides higher viscosity , has a strong binding ability to metal ions, microbial cells themselves and protein particles. Microbial resources in deep-sea hydrothermal vents are considered to be important resources capable of secreting new polysaccharides with special functions. Because, on the one hand, the special structure of exopolysaccharides secreted by deep-sea hydrothermal vent microorganisms indicates that it has new potential biological functions and will have new uses in the field of biotechnology; Exopolysaccharides can be used as model materials to study the stability and protection of biological macromolecules in extreme environments. Therefore, the study of deep-sea microbial polysaccharides has increasingly attracted the attention of scientists from various countries, and has become a research hotspot in the field of microbiology. At the same time, it has also become the focus of competition among various countries. my country should strengthen the research on deep-sea microbial polysaccharides, so as to occupy a place in this high-tech field of marine biology.

Microbial polysaccharides are important biotechnology products, and have a wide range of applications in the fields of medicine, biotechnology, food, environmental protection, and cosmetics. There are many types of microbial polysaccharides, and the structure of polysaccharides determines their functions. Due to its unique extreme ecological characteristics such as high pressure, high salinity, and low temperature, the exopolysaccharides secreted by deep-sea microorganisms should have new structural properties. In the past, more attention was paid to the structure of exopolysaccharides secreted by microorganisms in deep-sea hydrothermal vents and their role in the adaptation of microorganisms to the extreme environment of hydrothermal vents. However, abyssal hydrothermal vents occupy a small area in the entire deep seabed, and most of the microorganisms growing in this area are thermophilic (resistant) bacteria or mesophilic bacteria. Most of the deep seabed is an environment of high pressure, low temperature, weak light, and oligotrophic seawater flow, and a large number of psychrophilic (suitable) microorganisms grow in these areas.

In 2001, Chen Xiulan et al. first reported the strain screening, characterization and identification of the deep-sea psychrotroph Pseudomonas sp.SM9913, see Chen Xiulan et al., "Low temperature protease produced by deep-sea psychrotroph SM9913", "Marine Science", 2001, No. 5 Vol. 1, pp. 4-8. Chinese patent 01127405.0 discloses a method for producing cold-adaptive flavor protease by using the strain. But so far, there have been no reports on the structure and function of exopolysaccharides of deep-sea cold-adapted microorganisms. However, the research on this issue has more general significance for the utilization of deep-sea microorganisms and is worthy of further exploration.

The invention provides a method for preparing exopolysaccharide of deep-sea cold-adapted microorganisms.

The present invention firstly provides a process for preparing exopolysaccharides through liquid submerged fermentation using deep-sea psychrotrophic bacteria Pseudomonas sp. Lay the groundwork for applications in the field of technology.

Technical scheme of the present invention is as follows:

A method for preparing exopolysaccharides from deep-sea cold-suitable microorganisms, comprising the steps of:

(1) Seed preparation

Culture medium: 2-3 parts of bean cake powder, 2-3 parts of corn, 1-1.5 parts of bran, _0.4-0.5 part of _Na2HPO4 , _0.03-0.04 part of _KH2PO4 and 100 parts of water, all by weight, The culture medium was sterilized at 100-120°C for 30-50 minutes, and after cooling, the deep-sea psychrogenic bacterium Pseudomonas sp.SM9913 was used as the strain (see Chen Xiulan et al., "The low-temperature protease produced by the deep-sea psychic bacterium SM9913", "Marine Science" 2001 On January 19, the strain holder Shandong University), received the bacterial suspension of the eggplant bottle strain, and the inoculum amount was 5-7% by mass. Ventilation, stirring, cultivation. Obtain the seed solution and set aside.

Preferably, 300-350 milliliters of soybean oil is added to the above medium as a defoamer.

Preferably, the above-mentioned seeds are prepared by loading 70 liters of samples in a 150-liter fermenter, at 10-12° C., ventilating 1:0.5-0.6, stirring at 320-330 rpm, and culturing for 36-48 hours. The exopolysaccharide yield was about 5 g/L.

(2) Preparation of exopolysaccharide fermentation broth by liquid submerged fermentation

Medium: 3-3.5 parts of bean cake powder, 2.5-3 parts of corn flour, 2.5-3 parts of potato flour, 2.0-2.5 parts of bran, 0.4-0.5 parts of Na ₂ HPO ₄ , 0.03-0.04 parts of KH ₂ PO ₄ and 100 parts of water are parts by weight. 1000 liters of fermenter, 450 liters of culture medium, pH 6.2～6.3 after sterilization, inoculate 5～7% of the seeds prepared in step (1), stir 200～220 rpm, control the ventilation rate 0-12 hours 1: 0.5-0.6, 12-20 hours 1: 0.6-0.7, 20-28 hours 1: 0.8-0.9, control the culture temperature at 10-12°C, and ferment for 48-72 hours. The amount of polysaccharide produced per liter can reach 5 grams.

Preferably, 1 to 1.2 kg of soybean oil is added to the above medium as a defoamer.

(3) Post-treatment of exopolysaccharide fermentation broth

The exopolysaccharide fermentation liquid is centrifuged at 10,000 rpm, and then the centrifuged supernatant is precipitated with absolute ethanol. The volume ratio of the supernatant to absolute ethanol is 1:3. After protein removal and decolorization, the crude polysaccharide is obtained by centrifugal drying. The crude polysaccharide is redissolved in hot water at 60-70°C, concentrated to 20-30% of the original volume, separated by gel filtration chromatography and ion exchange chromatography, and vacuum freeze-dried to obtain the pure polysaccharide.

Finally, after quality inspection, it is packaged into exopolysaccharide products.

The making of the eggplant bottle slant bacterial classification described in the above-mentioned steps (1) can be by prior art. The present invention provides following specific operation steps:

Use 0.2-0.4 parts of beef extract, 0.8-1.2 parts of peptone, 1.5-2 parts of agar, 0.4-0.6 parts of NaCl as the medium, 95-100 parts of water, pH 7.0-7.2, sterilize, and scratch on the inclined surface of the test tube. Connect the deep-sea psychrotrophic bacteria Pseudomonassp.SM9913 strain, culture at 12-15°C for 24-26 hours, and store at 4°C.

Transfer the above strains into the slant of the eggplant bottle (the culture medium is the same as that of the preserved slant), incubate at 12-15°C for 24-26 hours, and use it as a bacterial suspension.

Preferred in above-mentioned steps (3) gel filtration chromatography and ion exchange chromatography:

Gel filtration chromatography column: 100cm×1.2cm, gel type: Sephadex G-100.

Ion exchange chromatography column: 25cm×1.6cm, gel type: DEAE-Sepharose Fast Flow.

The present invention will be further described below in conjunction with the structural analysis of exopolysaccharides.

The pure polysaccharide prepared by the method of the present invention undergoes 1D, 2D-NMR, MS, and methylation analysis to obtain the main structural composition repeating unit of the polysaccharide:

{→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

Among them, sugar unit: acetyl group: ethanol group = 4:5:1, highly acetylated α-(1→6) constitutes the core structure (61.8%) of the polysaccharide main chain, and there is a small amount of Ara (11.0%) at the end of the polysaccharide chain. %), Xyl (3.9%), Gal (3.1%), (1→4)Glc (5%) and (3→6)Glc (4%). From the exopolysaccharide structure, it can be seen that high acetylation can change the orientation and lateral order of polysaccharide molecules, thereby changing the physical properties of polysaccharides. The introduction of acetyl groups changes the stretching of molecules, which eventually leads to the exposure of polysaccharide hydroxyl groups. Increase its solubility in water, so as to achieve the effect of improving its biological activity.

The prepared pure polysaccharide of the present invention is a biotechnology product, and has wide applications in the fields of medicine, biotechnology, food, environmental protection, cosmetics and the like.

The technical characteristics of the present invention are:

1. The ocean is a special extreme environment, and the EPS secreted by marine microorganisms should have a special structure and function, which has increasingly attracted the attention of scientists from all over the world. In recent years, many studies have been carried out on the structure and function of EPS secreted by mesophilic and heat-resistant microorganisms in deep-sea hydrothermal vents and polar cold-adapted microorganisms, and a variety of EPS with new structures and functions have been discovered. However, there has been no report on the structure and function of EPS secreted by deep-sea cold-adapted microorganisms. Because more than 60% of the environment in the world is a deep sea with a depth of more than 2000 meters, most of which are low temperature environments.

2. For the first time, the deep-sea psychrotroph Pseudomonas sp.SM9913 was used as the strain to ferment and produce exopolysaccharide, and the optimized fermentation production process of exopolysaccharide EPS synthesized and secreted by the strain was established.

3. For the first time, provide a technical method for the efficient separation and purification of extracellular polysaccharides secreted by deep-sea psychrotrophic bacteria Pseudomonas sp.SM9913, using nuclear magnetic resonance, infrared spectroscopy, mass spectrometry and other techniques to analyze its physical and chemical characteristics, analyze its structure, and the main structural composition of polysaccharides As mentioned above, this is a new structure of deep-sea microbial exopolysaccharide.

Detailed ways

The present invention will be further described below in conjunction with the examples, but not limited thereto.

Embodiment 1: the preparation of the bacterial suspension of eggplant bottle bacterial classification

Use 0.3 parts of beef extract, 1.0 parts of peptone, 1.7 parts of agar, 0.5 parts of NaCl as the medium, 96 parts of water, pH 7.0 to 7.2, sterilize, and streak the deep-sea psychrotroph Pseudomonas sp.SM9913 on the inclined surface of the test tube Strain (see Chen Xiulan et al., "Low-temperature protease produced by deep-sea psychrotroph SM9913", "Marine Science" January 19, 2001, strain holder Shandong University), cultivated at 12-15°C for 25 hours, and stored at 4°C . Move the strain into the slant of the eggplant bottle (the culture medium is the same as that of the preserved slant), and cultivate it at 12-15°C for 25 hours, and use it as a bacterial suspension.

Embodiment 2: The preparation of exopolysaccharide of deep-sea cold-adapted microorganisms, the steps are as follows:

(1) Seed preparation

Culture medium: 2 parts of bean cake powder, 3 parts of corn, 1.5 parts of bran, 0.5 parts of Na ₂ HPO ₄ , 0.04 parts of KH ₂ PO ₄ and 100 parts of water, all by weight, add 300 ml of soybean oil to the medium for defoaming agent. Substrate was sterilized at 120° C. for 50 minutes, and after cooling, 70 liters of samples were loaded in a 150 liter fermenter, and the bacterial suspension of the eggplant bottle bacterial classification prepared in Example 1 was connected, and the inoculum size was 5%. At 10-12°C, ventilate 1:0.5-0.6, stir at 320-330 rpm, and cultivate for 40 hours.

(2) Preparation of exopolysaccharide fermentation broth by liquid submerged fermentation

Medium: 3.5 parts of bean cake powder, 2.5 parts of corn flour, 3 parts of potato flour, 2.5 parts of bran, 0.4 part of Na ₂ HPO ₄ , 0.03 part of KH ₂ PO ₄ and 100 parts of water, all in parts by weight. Add 1 kg of soybean oil to the culture medium as a defoamer. 1000 liters of fermenter, 450 liters of culture medium, pH 6.2～6.3 after sterilization, seed prepared in inoculation step (1), inoculum size 5%, stirring 220 rpm, control ventilation rate 0-12 hours 1: 0.5 ～0.6, 12-20 hours 1: 0.6～0.7, 20-28 hours 1: 0.8～0.9, control culture temperature 10～12 ℃, ferment for 56 hours. The amount of polysaccharide produced per liter can reach 5 grams.

(3) Post-treatment of exopolysaccharide fermentation broth

The exopolysaccharide fermentation broth was centrifuged at 10,000 rpm, then precipitated with absolute ethanol (the volume ratio of centrifuged supernatant to absolute ethanol was 1:3), deproteinized and decolorized, and then centrifuged and dried to obtain a crude product of polysaccharide. The crude polysaccharide is redissolved in hot water at 60-70°C, concentrated to 22% of the original volume, separated by gel filtration chromatography and ion exchange chromatography, and vacuum freeze-dried to obtain a pure polysaccharide (solid). Gel filtration chromatography and ion exchange chromatography are preferred: gel filtration chromatography column: 100cm×1.2cm, gel type: Sephadex G-100; ion exchange chromatography column: 25cm×1.6cm, gel type: DEAE -Sepharose Fast Flow.

The prepared pure polysaccharide is analyzed by 1D, 2D-NMR, MS, and methylation to obtain the main structural composition repeating unit of this polysaccharide:

{→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

Among them, sugar unit: acetyl group: ethanol group = 4:5:1, highly acetylated α-(1→6) constitutes the core structure (61.8%) of the polysaccharide main chain, and there is a small amount of Ara (11.0%) at the end of the polysaccharide chain. %), Xyl (3.9%), Gal (3.1%), (1→4)Glc (5%) and (3→6)Glc (4%).

Example 3:

A method for preparing exopolysaccharides from deep-sea cold-suitable microorganisms, comprising the steps of:

(1) Seed preparation

Culture medium: 2 parts of bean cake powder, 3 parts of corn, 1.5 parts of bran, 0.5 parts of Na ₂ HPO ₄ , 0.04 parts of KH ₂ PO ₄ and 100 parts of water, all by weight, add 300 ml of soybean oil to the medium for defoaming agent. The culture medium was sterilized at 120° C. for 50 minutes, and after cooling, inoculate the bacterial suspension (which may be the product of Example 1) of the SM9913 strain eggplant bottle strain with an inoculation amount of 5% by mass. The above-mentioned seed preparation is to load 70 liters of samples in a 150-liter fermenter, at 10-12 ° C, ventilate 1: 0.5-0.6, stir at 320-330 rpm, and cultivate for 48 hours. Exopolysaccharide production was 5 g/L.

(2) Preparation of exopolysaccharide fermentation broth by liquid submerged fermentation

Culture medium: 3.5 parts of bean cake powder, 2.5 parts of corn flour, 3 parts of potato flour, 2.5 parts of bran, 0.4 part of Na ₂ HPO ₄ , 0.03 part of KH ₂ PO ₄ and 100 parts of water, all are parts by weight. 1.2 kg of soybean oil was used as a defoamer. 1000 liters of fermenter, 450 liters of culture medium, pH 6.2～6.3 after sterilization, inoculate the seeds prepared in step (1), inoculum size 6%, stir 200～220 rpm, control the ventilation rate 0-12 hours 1 : 0.5-0.6, 12-20 hours 1: 0.6-0.7, 20-28 hours 1: 0.8-0.9, control the culture temperature at 10-12°C, and ferment for 72 hours. The amount of polysaccharide produced per liter can reach 5 grams.

(3) Post-treatment of exopolysaccharide fermentation broth

The exopolysaccharide fermentation broth was centrifuged at 10,000 rpm, then precipitated with absolute ethanol (the volume ratio of centrifuged supernatant to absolute ethanol was 1:3), deproteinized and decolorized, and then centrifuged and dried to obtain a crude product of polysaccharide. The crude polysaccharide is redissolved in hot water at 65°C, concentrated to 30% of the original volume, separated by gel filtration chromatography and ion exchange chromatography, and vacuum freeze-dried to obtain the pure polysaccharide. Gel filtration chromatography and ion exchange chromatography are preferred: gel filtration chromatography column: 100cm×1.2cm, gel type: Sephadex G-100; ion exchange chromatography column: 25cm×1.6cm, gel type: DEAE -Sepharose Fast Flow.

The polysaccharide structure composition is the same as that in Example 2.

Claims (7)

1. A method for preparing exopolysaccharides from deep-sea cold-adapted microorganisms, comprising the steps of: (1) Seed preparation Culture medium: 2-3 parts of bean cake powder, 2-3 parts of corn, 1-1.5 parts of bran, _0.4-0.5 part of _Na2HPO4 , _0.03-0.04 part of _KH2PO4 and 100 parts of water, all by weight, Sterilize the culture medium at 100-120°C for 30-50 minutes, after cooling, use the deep-sea psychrogenic bacterium Pseudomonas sp.SM9913 as the strain, connect the bacterial suspension of the eggplant bottle strain, the inoculation amount is 5-7% by mass; ventilate and stir , cultivated; the seed liquid was obtained and set aside; (2) Preparation of exopolysaccharide fermentation broth by liquid submerged fermentation Medium: 3-3.5 parts of bean cake powder, 2.5-3 parts of corn flour, 2.5-3 parts of potato flour, 2.0-2.5 parts of bran, 0.4-0.5 parts of Na ₂ HPO ₄ , 0.03-0.04 parts of KH ₂ PO ₄ and 100 parts of water are all parts by weight; 1000 liters of fermenter, 450 liters of culture medium, pH6.2～6.3 after sterilization, inoculate 5～7% of the seeds prepared in step (1), stir 200～220 rpm, Control ventilation for 0-12 hours 1: 0.5-0.6, 12-20 hours 1: 0.6-0.7, 20-28 hours 1: 0.8-0.9, control culture temperature 10-12°C, ferment for 48-72 hours; (3) Post-treatment of exopolysaccharide fermentation broth The exopolysaccharide fermentation liquid is centrifuged at 10,000 rpm, and then the centrifuged supernatant is precipitated with absolute ethanol. The volume ratio of the supernatant to absolute ethanol is 1:3. After protein removal and decolorization, the crude polysaccharide is obtained by centrifugal drying. The crude polysaccharide is redissolved in hot water at 60-70°C, concentrated to 20-30% of the original volume, separated by gel filtration chromatography and ion exchange chromatography, and vacuum freeze-dried to obtain the pure polysaccharide. 2. The method for preparing exopolysaccharides from deep-sea cryogenic microorganisms according to claim 1, characterized in that 300-350 ml of soybean oil is added to the culture medium in step (1) as a defoamer. 3. The method for preparing exopolysaccharides from deep-sea cold-adapted microorganisms according to claim 1, characterized in that the seed preparation in step (1) is carried out in a 150-liter fermenter with a sample volume of 70 liters at 10-12°C , ventilation 1: 0.5-0.6, stirring at 320-330 rpm, and culturing for 36-48 hours. 4. The method for preparing exopolysaccharide of deep-sea cold-adapted microorganisms as claimed in claim 1, characterized in that, the specific steps of making the eggplant bottle slant strain described in step (1) are as follows: Use 0.2-0.4 parts of beef extract, 0.8-1.2 parts of peptone, 1.5-2 parts of agar, 0.4-0.6 parts of NaCl as the medium, 95-100 parts of water, pH 7.0-7.2, sterilize, and scratch on the inclined surface of the test tube. Connect the deep-sea psychrotroph Pseudomonassp.SM9913 strain, culture at 12-15°C for 24-26 hours, store at 4°C; transfer the above-mentioned strains into the slant of the eggplant bottle, the medium is the same as that of the preserved slant, and culture at 12-15°C for 24-24 hours. For 26 hours, it is used as a bacterial suspension. 5. The method for preparing exopolysaccharides from deep-sea cold-adapted microorganisms according to claim 1, characterized in that 1-1.2 kg of soybean oil is added to the culture medium in step (2) as a defoamer. 6. The method for preparing exopolysaccharides from deep-sea cold-adapted microorganisms according to claim 1, wherein the gel filtration chromatography column in step (3) is: 100cm×1.2cm, Sephadex G -100. 7. The method for preparing extracellular polysaccharides from deep-sea cold-adapted microorganisms according to claim 1, characterized in that the ion-exchange chromatography in step (3) uses an ion-exchange chromatography column with a size of 25cm×1.6cm and DEAE-Sepharose Fast Flow.