CN102174614B - Antarctic cold-adapted microbial extracellular polysaccharide capable of improving body immunity - Google Patents

Abstract

The invention relates to an exopolysaccharide of Antarctic cold-adapted microorganisms capable of improving body immunity, and belongs to the technical field of medicine and health food. The present invention prepares cold-adaptive microbial exopolysaccharides by cultivating, fermenting, separating and purifying Antarctic cold-adapted bacteria (Pseudoaltermonassp.) S-5 with strain preservation number CGMCC NO.3433. Its molecular weight is 39,7046 Da, and it is composed of mannose, glucose and galactose in a molar ratio of 4.8:50.9:44.3. The exopolysaccharide of Antarctic cold-adapted microorganisms prepared by the invention, which can improve the immunity of the body, can promote the proliferation of spleen lymphocytes in vitro, enhance the activity of macrophages, and improve the immunity of the body.

Description

An exopolysaccharide from an Antarctic cold-adapted microorganism that can improve the body's immunity

technical field

The invention relates to an exopolysaccharide of Antarctic cold-adapted microorganisms capable of improving body immunity, and belongs to the technical field of medicine and health food.

Background technique

The Antarctic ocean is covered with ice and snow all year round, and the geographical environment and climate characteristics are very special. The microorganisms living in this extreme environment have a high degree of diversity. This diversity provides abundant resources for the development of active substances with special application value. The exopolysaccharide produced by Antarctic microorganisms can alleviate the damage of low temperature and high salt to the bacteria, and is a good antifreeze protectant, which plays an important role in low temperature protection for microorganisms to adapt to the harsh environment of Antarctic low temperature and high salt. The exopolysaccharides of Antarctic microorganisms have novel structures and unique functions, and are important resources for the development of new polysaccharide immunomodulators. For example, the exopolysaccharide produced by the Antarctic microorganism Pseudoaltermonas sp. S-15-13 is a mannan with a molecular weight of 62kDa. This exopolysaccharide has certain immune activity. (Production and characterization of anextracellular polysaccharide of antarctic marine bacteria Pseudoalteromonas sp.S-15-13; LI Jiang, CHEN Kaoshan, LIN Xuezheng, HE Peiqing, LI Guangyou; International standard dry number: 0253-505X; 2006.6).

Antarctic cold-adapted bacteria Pseudoal termonas sp.S-5 belongs to the genus Pseudoalteromonas. The bacterium is rod-shaped, and the physiological and biochemical identification results show that the bacterium is Gram-staining negative, facultative anaerobic, oxidase, catalase, urease, lysine decarboxylase, arginine decarboxylase, arginine double The hydrolase reaction was positive; the nitrate reduction reaction, indole test, gelatin liquefaction test, H2R test, MR test, VP test, ONPG test, ornithine desustase reaction were all negative.

The fermented liquid of Antarctic cold-adapted bacterium Pseudoal termonas sp.S-5 can be applied to the anti-cold aspect of plants and crops, such as the Chinese patent whose publication number is CN101775361A (application number 200910256034.6), in which the antifreeze active substance is Antarctic cold-adapted The exopolysaccharide produced by the bacterium Pseudoaltermonas sp.S-5 has a molecular weight of about 39,7046Da, and the efficacy of this exopolysaccharide in improving the body's immunity has not been reported.

Contents of the invention

Aiming at the deficiencies of the prior art, the invention provides an exopolysaccharide of Antarctic cold-adapted microorganisms capable of improving the body's immunity and its application in improving the body's immunity.

A method for preparing an exopolysaccharide of cold-adapted microorganisms in Antarctica capable of improving body immunity, characterized in that it comprises the following steps:

(1) After activating the Antarctic cold-adapted bacteria (Pseudoaltermonas sp.) S-5 with the strain preservation number CGMCC NO.3433, inoculate it in Zobell 2216E liquid medium, cultivate it at 6-10°C for 22-25 hours, press 1 -2wt% was inoculated in Zobell 2216E liquid medium for expanded culture to obtain the multiplied strains;

(2) Inoculate the fermentation medium in the liquid fermenter with the inoculum amount of 8-12wt% by the multiplication bacterial classification that step (1) makes, 7-9 ℃ of aerobic stirring culture 50-60h, stirring speed 150 -180r/min, to get liquid fermentation broth;

(3) Centrifuge the liquid fermentation broth prepared in step (2) for 15min at 12000r/min, and collect the supernatant;

(4) Concentrate the supernatant obtained in step (3) to 1/4 of the original volume to obtain a concentrated solution, then add three times the volume of 95% ethanol to the concentrated solution, precipitate overnight, centrifuge, and collect the precipitate;

(5) dissolving the precipitate collected in step (4) with water to obtain a solution, and then adding seveage reagent to remove protein to obtain a crude sugar solution;

(6) After the crude sugar solution prepared in step (5) is decolorized by macroporous resin D-301R, it is separated through a DEAE-Fast Flow anion exchange column and a Sephadex G-75 chromatographic column to obtain a purified exopolysaccharide solution;

(7) Vacuum freeze-drying the exopolysaccharide solution prepared in step (6) to prepare exopolysaccharides from Antarctic cold-adaptive microorganisms that can improve the body's immunity.

The components of the Zobell 2216E liquid medium in the step (1) are as follows, all in percentage by weight: 0.5% peptone, 0.1% yeast powder, 2% glucose, the remainder of artificial seawater, pH 7.0.

The fermentation medium components in the step (2) are as follows, all in percentage by weight: 0.5% peptone, 0.1% yeast powder, 2% glucose, 2% sodium chloride, artificial seawater balance, pH7.0.

Before the stirring culture in the step (2), it also includes adding 0.3% sterilized soybean oil by weight of the fermentation medium to the fermentation medium. Sterilized soybean oil was used as a defoamer to eliminate air bubbles generated during stirring.

The centrifugation condition in the step (3) is: 12000r/min, 15min.

The concentration conditions in the step (4) are: 60° C., rotary evaporation; the concentrated solution is about 1/4 of the volume of the supernatant.

The seveage reagent in the step (5) is composed of chloroform and n-butanol in a volume ratio of 4:1; the amount of the seveage reagent added is 1/3 of the volume of the solution.

The artificial seawater is prepared from Instant Ocean Reef Crystals seawater salt.

The exopolysaccharide of Antarctic cold-adapted microorganisms prepared by the above-mentioned preparation method and capable of improving the body's immunity has a molecular weight of 39,7046 Da and is composed of mannose, glucose and galactose in a molar ratio of 4.8:50.9:44.3.

The application of the exopolysaccharide of cold-adapted microorganisms in the preparation of drugs and health products for promoting the proliferation of spleen lymphocytes in vitro, enhancing the activity of macrophages and improving the body's immunity.

Beneficial effect

The exopolysaccharide of Antarctic cold-suitable microorganisms prepared by the preparation method of the invention and capable of improving body immunity can promote the proliferation of spleen lymphocytes in vitro, enhance the activity of macrophages, and improve body immunity.

Description of drawings

Fig. 1 is the infrared spectrum detection collection of cold-adapted microorganism exopolysaccharide;

Fig. 2 is the high-performance permeation chromatogram detection collection of exopolysaccharides of cold-adapted microorganisms;

Fig. 3 is the gas chromatographic detection spectrum of exopolysaccharide of cold-adapted microorganism;

In the figure: a, mannose, b, glucose, c, galactose, d, inositol hexaacetyl ester.

Detailed ways

The cold-adapted bacteria S-5 (Pseudoaltermonas sp.S-5) described in the examples has been preserved in the General Microbiology Center of China Committee for the Collection of Microbial Cultures on November 9, 2009, address: Datun Road, Chaoyang District, Beijing , Institute of Microbiology, Chinese Academy of Sciences, the preservation number is: CGMCC NO.3433.

The Instant Ocean Reef Crystals seawater salt described in the examples was purchased from Shanghai Yujie Aquarium Products Co., Ltd.

Example 1

A method for preparing an exopolysaccharide of cold-adapted microorganisms in Antarctica capable of improving body immunity, comprising the steps of:

(1) After activating the Antarctic cold-adapted bacterium (Pseudoaltermonnas sp.) S-5 with the strain preservation number CGMCC NO.3433, inoculate it in Zobell 2216E liquid medium, cultivate it at 9°C for 24 hours, and inoculate it at 1wt% in Zobell 2216E liquid medium expands the culture, and the bacteria can be multiplied;

(2) by the inoculum amount of 10wt% by the multiplication bacterial classification that step (1) makes, be inoculated in the fermentation medium in the liquid fermentation tank, after adding the sterilized soybean oil of fermentation medium weight 0.3% in the fermentation medium , 7-9°C aerobic stirring culture for 50-60h, stirring speed 160-170r/min, to obtain a liquid fermentation broth;

(3) Centrifuge the liquid fermentation broth prepared in step (2) for 15min at 12000r/min, and collect the supernatant;

(4) Concentrate the supernatant obtained in step (3) to 1/4 of the original volume by rotary evaporation at 60°C to obtain a concentrated solution, then add three times the volume of 95% ethanol to the concentrated solution overnight Precipitate, centrifuge, collect the precipitate;

(5) Dissolve the precipitate collected in step (4) with water, add 1/3 volume of seveage reagent (chloroform: n-butanol volume ratio is 4: 1) to remove protein, and obtain a crude sugar solution;

(6) Decolorize the crude sugar solution prepared in step (5) through macroporous resin D-301R, and then separate it through DEAE-Fast Flow anion exchange column and Sephadex G-75 chromatography column to obtain a purified exopolysaccharide solution ;

(7) Vacuum freeze-drying the exopolysaccharide solution prepared in step (6) to prepare exopolysaccharides from Antarctic cold-adaptive microorganisms that can improve the body's immunity.

The components of the Zobell 2216E liquid medium in the step (1) are as follows, all in percentage by weight: 0.5% peptone, 0.1% yeast powder, 2% glucose, the remainder of artificial seawater, pH 7.0.

The components of the fermentation medium in the step (2) are as follows, all in percent by weight: 2% glucose, 2% sodium chloride, the remainder of artificial seawater, pH 7.0.

The artificial seawater is prepared from Instant Ocean Reef Crystals seawater salt.

Product properties:

The exopolysaccharide of Antarctic cold-adapted microorganisms prepared by the above method, which can improve the body's immunity, is a solid dry powder in the form of flocculents. The iodine-potassium iodide reaction, biuret reaction, Molish reaction, and ultraviolet detection show that there is no protein or nucleic acid in the sample. Such impurities exist, and through infrared spectrum detection, it is found that the characteristic peak absorption of polysaccharides proves that it is a polysaccharide (as shown in Figure 1); by high performance permeation chromatography, its molecular weight is 39,7046Da (as shown in Figure 2); The monosaccharide composition measured by gas chromatography is mannose:glucose:galactose=4.8:50.9:44.3 (as shown in Figure 3), indicating that this exopolysaccharide is a heteropolysaccharide.

Example 2 Effect of in vitro administration of exopolysaccharides from cold-adapted microorganisms on the active proliferation of mouse spleen lymphocytes

The mice were killed by cervical dislocation, and the spleen was aseptically removed to prepare splenic lymphocytes, and a 1×10 ⁶ /ml cell suspension was prepared with RPMI 1640 culture medium containing 10% fetal bovine serum. Add mouse 1×10 ⁶ /ml splenocyte suspension into a 96-well cell culture plate, 100 μl per well, set up a control group, exopolysaccharide (final concentrations of 0.1, 0.25, 0.5, 1, 2.5, 5, 10 μg/ml) stimulation group and ConA (final concentration: 5 μg/ml) stimulation group, each group had six replicate wells for each concentration, and the blank control group was replaced by an equal volume of RPMI 1640. Incubate for 72 hours at 37°C in a 5% CO ₂ saturated humidity incubator. 10 μl of MTT solution was added to each well 4 hours before the end of the culture, and the culture was continued until the end of the experiment. Take it out, suck off 100 μl of the supernatant, add 100 μl of dimethyl sulfoxide (DMSO) to each well to dissolve formazan, and read the OD value of each well at a wavelength of 570 nm on a microplate reader.

The cold-adapted microbial exopolysaccharide prepared in Example 1 can promote the proliferation activity of mouse spleen lymphocytes in vitro, and the experimental results are shown in Table 1. Exopolysaccharides from cold-adapted microorganisms can significantly promote the proliferation of splenic lymphocytes. Lymphocyte proliferation can be significantly promoted in the concentration range of 0.1-10 μg/ml (P<0.01), the optimum concentration is 0.5 μg/ml, and the light absorption value is increased by 39.7% compared with the control group.

Table 1 Effects of exopolysaccharides from cold-adapted microorganisms on the proliferation activity of mouse spleen lymphocytes in vitro (n=6)

*Indicates that after the t test, there is a significant difference compared with the control group (P<0.05)

** indicates that after t test, there is a very significant difference compared with the control group (P<0.01)

Example 3: Influence of in vitro administration of exopolysaccharides from cold-adapted microorganisms on the activity of acid phosphatase in mouse macrophages

Mice were intraperitoneally injected with 1 ml of 4% starch solution, and killed by cervical dislocation 3 days later. Aseptically collected mouse peritoneal macrophages to prepare cell suspension; the peritoneal cell suspension was added to a 96-well cell culture plate, 100 μl per well. Cultivate in a CO ₂ incubator at 37°C and 5% CO ₂ for 4 hours. Remove the supernatant, and the adherent cells are macrophages. The experiment set up control group, extracellular polysaccharide (0.1, 0.25, 0.5, 1, 2.5, 5, 10 μg/ml final concentration) stimulation group, LPS (1 μg/ml final concentration) stimulation group, 100 μl per hole, each group Six replicate wells were set up for each concentration. Cultivate in a CO ₂ incubator at 37°C and 5% CO ₂ for 24 hours. Take it out, remove the supernatant, add 100 μl of pNPP solution to each well, mix well, place at 37°C for 30 min, take it out, and immediately add 10 μl of 1.0mol/L NaOH solution to stop the reaction. Read the OD value of each well at a wavelength of 405 nm on a microplate reader. To detect the effect of in vitro administration of exopolysaccharides from cold-adapted microorganisms on the activity of acid phosphatase in mouse macrophages.

The cold-adapted microbial exopolysaccharide prepared in Example 1 stimulated the acid phosphatase activity of mouse peritoneal macrophages in vitro, and the experimental results are shown in Table 2. Exopolysaccharides from cold-adapted microorganisms can significantly enhance the activity of macrophage acid phosphatase in the concentration range of 0.1-10 μg/ml, and with the increase of the concentration, the effect is gradually enhanced in a dose-dependent manner. And when the concentration is 10 μg/ml, the activity of macrophage acid phosphatase is increased by 302.7% compared with the control group.

Table 2 Effects of exopolysaccharides from cold-adapted microorganisms on the activity of acid phosphatase in mouse macrophages (n=6)

*Indicates that after the t test, there is a significant difference compared with the control group (P<0.05)

** indicates that after t test, there is a very significant difference compared with the control group (P<0.01)

Example 4: In vivo administration of exopolysaccharides from cold-adapted microorganisms to the immune regulation of normal mice

50 rats (with a body weight of 18-22g) were randomly divided into 5 groups, 10 in each group, respectively a control group and an administration group, wherein the dosage of the administration group was respectively the cold-adaptive microorganism exopolysaccharide prepared in Example 1 1.25, 2.5, 5, 10mg/kg/day, continuous gavage for 10 days, detect the influence on the spleen index, thymus index, and macrophage phagocytic activity of normal mice.

Determination of macrophage phagocytosis activity On the seventh day of the experiment, mice were sensitized by intraperitoneal injection of 1 ml of 4% starch solution. On the 10th day of the experiment, each mouse was injected with 1ml of 1% chicken red blood cell saline solution, and after 30 minutes, the eyeballs were removed to take blood, and the abdominal cavity was dissected after being killed, and the peritoneal exudate was sucked, and the peritoneal fluid was placed on a glass slide, smeared, fixed, and smeared. Stained with Shi's staining solution, rinsed, dried, observed under a microscope, and calculated the phagocytosis percentage and phagocytosis index.

Phagocytosis percentage: the number of macrophages that phagocytized chicken red blood cells per 100 macrophages.

Phagocytosis index: the total number of chicken erythrocytes phagocytosed per 100 macrophages divided by 100, that is, the average number of chicken erythrocytes phagocytosed by each macrophage.

The experimental results are shown in Table 3. In vivo administration of exopolysaccharides from cold-adapted microorganisms can significantly affect the phagocytosis rate and phagocytosis index of mouse macrophages phagocytizing chicken red blood cells. Compared with the control group, there is a very significant difference (P<0.01). When the concentration is 10mg/kg/day, the phagocytosis rate is 62.08% higher than that of the control group, and the phagocytosis index is 103.09% higher than that of the control group. Cell activity, thereby improving the non-specific immunity of mice.

Table 3 Effects of exopolysaccharides from live cold microorganisms on the phagocytic activity of normal mouse macrophages (n=10)

*Indicates that after the t test, there is a significant difference compared with the control group (P<0.05)

** indicates that after t test, there is a very significant difference compared with the control group (P<0.01)

Determination of organ coefficients On the 10th day of the experiment, the mice were weighed and sacrificed, and the spleen and thymus were taken after dissection, and the spleen index and thymus index were calculated respectively.

Spleen index (%)=(spleen weight/body weight)×100%

Thymus index (%) = (thymus weight/body weight) × 100%

The experimental results are shown in Table 4. The exopolysaccharide concentration of cold-adapted microorganisms at 1.25, 2.5, 5, and 10 mg/kg/day can significantly affect the thymus index of mice. When the exopolysaccharide concentration of cold-adapted microorganisms is 10 mg/kg/day, the spleen index is higher than that of the control group 28.57%, the thymus index increased by 166.67% compared with the control group, and there was a very significant difference compared with the control group (P<0.01), which indicated that the exopolysaccharide of cold-adapted microorganisms could promote the growth of immune organs in mice.

Table 4 Effects of exopolysaccharides from cold-adapted microorganisms on organ coefficients of normal mice (n=10)

*Indicates that after the t test, there is a significant difference compared with the control group (P<0.05)

** indicates that after t test, there is a very significant difference compared with the control group (P<0.01)

Claims (2)

1. A preparation method for an exopolysaccharide of cold-adapted microorganisms in Antarctica capable of improving immunity, characterized in that it comprises the steps: (1) Activate Pseudoaltermonas sp. S-5 with strain preservation number CGMCC NO.3433, inoculate in Zobell 2216E liquid medium, culture at 9°C for 24 hours, and inoculate at 1 wt% Expanded culture in Zobell 2216E liquid medium to obtain multiplied strains; (2) Inoculate the multiplied strains prepared in step (1) into the fermentation medium in the liquid fermenter at an inoculum amount of 10 wt%, and add 0.3% of sterilized soybean oil by weight of the fermentation medium into the fermentation medium , 7-9 ℃ aerobic stirring culture for 50-60h, stirring speed 160-170r/min, to obtain a liquid fermentation broth; (3) Centrifuge the liquid fermentation broth prepared in step (2) at 12000 r/min for 15 minutes, and collect the supernatant; (4) Concentrate the supernatant obtained in step (3) to 1/4 of the original volume by rotary evaporation at 60°C to obtain a concentrated solution, then add three times the volume of 95% ethanol to the concentrated solution overnight Precipitate, centrifuge, collect the precipitate; (5) Dissolve the precipitate collected in step (4) with water, add 1/3 volume of seveage reagent to remove protein, and obtain a rough sugar solution; (6) The crude sugar solution prepared in step (5) is decolorized by macroporous resin D-301R, and then separated by DEAE-Fast Flow anion exchange column and Sephadex G-75 chromatography column to obtain a purified exopolysaccharide solution ; (7) Vacuum freeze-drying the exopolysaccharide solution prepared in step (6) to obtain a pure exopolysaccharide from cryogenic microorganisms; The components of the Zobell 2216E liquid medium in the step (1) are as follows, all in percent by weight: peptone 0.5%, yeast powder 0.1%, glucose 2%, artificial seawater balance, pH7.0; The components of the fermentation medium in the step (2) are as follows, all in percent by weight: 0.5% peptone, 0.1% yeast powder, 2% glucose, 2% sodium chloride, artificial seawater balance, pH7.0; The artificial seawater is prepared from Instant Ocean Reef Crystals seawater salt. 2. The exopolysaccharide of Antarctic cold-adapted microorganisms that can improve the immunity of the body obtained by the preparation method of claim 1 has a molecular weight of 39,7046 Da, and is composed of mannose, glucose and galactose in a molar ratio of 4.8:50.9:44.3 constitute. 3. The application of the exopolysaccharide of Antarctic cold-adapted microorganisms as claimed in claim 2 in the preparation of drugs and health care products for promoting the proliferation of splenic lymphocytes in vitro, enhancing the activity of macrophages, and improving the immunity of the body.

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