阅读说明：本技术 一种具有免疫调节活性的小球藻胞外多糖复合物及其制备方法和应用 (Chlorella extracellular polysaccharide compound with immunoregulatory activity and preparation method and application thereof ) 是由 李玉芹 雷铮宇 周蓉 唐裕芳 祝佳惠 李超 贾淑婷 于 2019-11-22 设计创作，主要内容包括：本发明公开了一种具有免疫调节活性的小球藻胞外多糖复合物及其制备方法和应用。本发明的胞外多糖复合物提取自小球藻Chlorella protothecoides CS-41发酵液,经过滤、离心、浓缩、醇沉、Sevage法除蛋白后,再通过阴离子交换层析和凝胶排阻色谱层析等分离纯化工序获得,所得多糖复合物为白色粉末,平均相对分子量为170kDa,由脂质和多糖组成,其中脂质含棕榈酸和硬脂酸,多糖由鼠李糖、木糖、甘露糖、葡萄糖组成。本发明的胞外多糖复合物组成明确,具备免疫调节作用,能显著降低炎症因子(ROS、iNOS、IL-6和TNF-α)水平,在临床及相关医疗保健领域有着广阔的应用前景。(The extracellular polysaccharide compound is extracted from Chlorella protothecoides CS-41 fermentation liquor, protein is removed through filtration, centrifugation, concentration, alcohol precipitation and Sevage methods, and then the extracellular polysaccharide compound is obtained through separation and purification procedures such as anion exchange chromatography and gel exclusion chromatography, wherein the obtained polysaccharide compound is white powder, has the average relative molecular weight of 170kDa and consists of lipid and polysaccharide, wherein the lipid contains palmitic acid and stearic acid, and the polysaccharide consists of rhamnose, xylose, mannose and glucose.)

1. A chlorella exopolysaccharide complex having immunomodulatory activity, comprising: the chlorella extracellular polysaccharide compound is white powder, has the molecular weight of 170kDa, and consists of 16.52% of lipid and 82.58% of polysaccharide in percentage by mass, wherein the lipid consists of palmitic acid (C16: 039.44%) and stearic acid (C18: 060.56%) in percentage by mass, and the polysaccharide component consists of 4 monosaccharides including rhamnose, xylose, mannose and glucose in percentage by mass, and the mole percentages are 10.22%, 46.19%, 17.98% and 25.62% respectively.

2. The method for preparing chlorella exopolysaccharide complexes with immunomodulatory activity of claim 1, wherein the method comprises the steps of: the extracellular polysaccharide compound is extracted from chlorella fermentation liquor and is obtained by separation and purification, and the acquisition of the chlorella fermentation liquor comprises the following steps:

(1) inoculating Chlorella protothecoides CS-41 to a solid culture medium for rejuvenation, then transferring to a liquid culture medium to obtain a primary seed solution, and then continuing transferring to a fresh liquid culture medium for amplification culture to obtain a secondary seed solution;

(2) transferring the secondary chlorella seed solution obtained in the step 1) to a fresh liquid culture medium, and continuously carrying out amplification culture under the conditions of salt stress and mixotrophic culture to obtain chlorella fermentation liquor.

3. The method for preparing chlorella exopolysaccharide complexes with immunomodulatory activity as claimed in claim 2, wherein the method comprises the steps of: in the step (1), the culture medium is a Basal culture medium, the solid culture and the first-stage seed liquid culture are mixotrophic culture, the illumination intensity is 2000Lux, and the illumination period is 12 h: performing dark alternate culture for 12h at 28 deg.C for 5 days; the secondary seed liquid culture is heterotrophic culture, the inoculation amount is 5%, the culture temperature is 28 ℃, and the culture period is 5 days.

4. The method for preparing chlorella exopolysaccharide complexes with immunomodulatory activity as claimed in claim 2, wherein the method comprises the steps of: in the step (2), the inoculation amount of the chlorella secondary seed solution is 1%, the used liquid culture medium is a Basal culture medium, the salt stress concentration is 10 per mill salinity, the illumination intensity is 2000Lux, and the light dark period is 8h illumination: the cultivation is carried out alternately in 16h of darkness and at the temperature of 28 ℃ and the rotation speed of 160rpm for 7 days.

5. Use of the chlorella exopolysaccharide complex of claim 1 for inhibiting inflammation.

6. The use according to claim 5, wherein said Chlorella exopolysaccharide complex significantly reduces the production of inflammatory factors ROS, iNOS, IL-6 and TNF- α.

Technical Field

The invention relates to the field of functional health-care food or biological medicine, in particular to a chlorella extracellular polysaccharide compound with immunoregulation activity and a preparation method and application thereof.

Background

Microalgae biodiesel has become a third generation sustainable biofuel recognized worldwide, and the development and application of microalgae biodiesel have important significance for solving the problems of shortage of fossil fuels and air pollution at present. However, the microalgae energy industry still faces the dilemma of high cost, is still in the starting stage and is difficult to be commercially applied. Improving the accumulation rate of oil in algal cells and increasing the oil yield is one of effective solutions for reducing the cost of microalgae biodiesel.

Around the growth and oil accumulation characteristics of algae cells at home and abroad, by changing environmental conditions such as temperature, illumination, nutritional conditions, salinity, metal ions and the like, great achievements are obtained in the research field of improving the oil yield of the microalgae cells, for example, the oil accumulation of the algae cells can be greatly promoted by adopting an exponential terminal-short time high temperature stress mode to culture Scenedesmus quadricauda (Scenedesmus quadratus), so that the lipid content and the yield of the algae cells respectively reach 33.5 percent and 23.2mg L^-1d^-1(environ. technol.,2016,37: 2649-; the oil yield of Chlorella (Chlorella sp.) can be increased to 15.67mg L by inducing the stress of low-phosphorus environment^-1d^-1(J.appl.Phycol.,2013,25: 311-318.); faizol Bux et al, using ferric ammonium citrate stress, increased the lipid content and lipid productivity of KJ671624 of Fulvox Fusarium (Ankistrodesmus falcatus) to 59.6% and 74.07mg L, respectively^-1d^-1(biochem, eng.j, 2015,94, 22-29). Although the oil production efficiency of algae cells is greatly improved through the regulation and control of culture modes and culture conditions, so that the yield of the algae cells is improved, the price of microalgae biodiesel is still too expensive compared with the price of petroleum (Biofuels,2010,1: 763-784). Fruit of Chinese wolfberryIn fact, microalgae not only accumulate oil in cells during their growth, but also secrete a variety of metabolites, such as polysaccharides, proteins, lipids, pigments, vitamins, inhibitory and stimulatory factors, etc., into the extracellular environment. Most of the secondary metabolites have biological activity and have great development and application prospects, wherein the microalgae polysaccharide and the microalgae polysaccharide complex have various types, also have important biological activities of oxidation resistance, tumor resistance, virus resistance, blood fat reduction, blood sugar reduction and the like, and show great application potential in the fields of medicine and clinic. The development and utilization of secondary microalgae metabolites and the acquisition of high-value active substances are important ways for reducing the price of microalgae diesel and accelerating the industrialization process of microalgae.

Chlorella contains abundant polysaccharides with anti-tumor activity, pathogenic bacteria resistance, virus infection resistance and immunity enhancement and compounds thereof, but the research on polysaccharide substances of Chlorella protothecoides is less at present, and only Mario and the like analyze the cell wall polysaccharide composition (Arch. Mikrobiol.,1973,92,227 one 233) and George and the like to optimize the extraction process of intracellular polysaccharides (Chin.J.Appl.Environ.biol.,2014,20(4):615 one 620), so far, no related reports on the application values of the original extracellular polysaccharides and the compounds thereof exist. The polysaccharide compound with the immunoregulatory activity is separated and prepared from chlorella protothecoides CS-41 fermentation liquor, so that the extraction and preparation of chlorella oil cannot be influenced, a product with a medical health care prospect can be obtained by high-valued utilization of extracellular metabolites, and the polysaccharide compound has important significance for comprehensive development of microalgae resources and reduction of microalgae oil cost.

Disclosure of Invention

The invention aims to obtain high-value byproducts of chlorella, provides a chlorella extracellular polysaccharide compound with immunoregulatory activity and a preparation method thereof, and also provides the biological activity application of the immunoregulatory polysaccharide.

The technical scheme of the invention is as follows:

the chlorella extracellular polysaccharide complex with the immunoregulatory activity is white powder, has the average relative molecular weight of 170kDa, and consists of 16.52% of lipid and 82.58% of polysaccharide in percentage by mass, wherein the lipid consists of 39.44% of palmitic acid (C16:0) and 60.56% of stearic acid (C18:0) in percentage by mass, and the polysaccharide component consists of 4 monosaccharides, namely rhamnose, xylose, mannose and glucose in percentage by mole, and is 10.22%, 46.19%, 17.98% and 25.62% respectively.

The preparation method of the chlorella exopolysaccharide compound with the immunoregulation activity comprises the following steps:

(1) inoculating Chlorella protothecoides CS-41 to a solid culture medium for rejuvenation, then transferring to a liquid culture medium to obtain a primary seed solution, and then continuing transferring to a fresh liquid culture medium for amplification culture to obtain a secondary seed solution;

(2) continuously carrying out amplification culture on the chlorella secondary seed liquid obtained in the step (1) under the conditions of fresh liquid culture medium and salt stress and mixotrophic culture to obtain chlorella fermentation liquid;

(3) collecting the supernatant of the fermentation liquor obtained in the step (2), precipitating with absolute ethanol, collecting the precipitate for redissolution, adding Sevage reagent into the obtained sample to remove protein, dialyzing, and freeze-drying to obtain crude extracellular polysaccharide of chlorella;

(4) separating the crude extracellular polysaccharide of the chlorella obtained in the step (3) by anion exchange chromatography, performing gradient elution by using a Tris-HCl buffer solution containing 0-1 mol/L NaCl, collecting a target elution peak, dialyzing and freeze-drying;

(5) separating the elution peak obtained in the step (4) by using a molecular exclusion chromatography, collecting elution components, dialyzing, and freeze-drying to obtain single-component polysaccharide, namely the chlorella extracellular polysaccharide with the immunoregulatory activity, which is named as CPEPS-2.

Further, in the step (1), the solid culture medium is a Basal culture medium, the solid culture and the first-level seed liquid culture are mixotrophic culture, the illumination intensity is 2000Lux, and the illumination period is 12h illumination: performing dark alternate culture for 12h at 28 deg.C for 5 days; the secondary seed liquid culture is heterotrophic culture, the inoculation amount is 5%, the culture temperature is 28 ℃, and the culture time is 5 days.

Further, in the step (2), the liquid culture medium is a Basal culture medium, the salt stress concentration is 10 per mill salinity, the inoculation amount is 1%, the illumination intensity is 2000Lux, and the illumination period is 8h illumination: the cultivation is carried out alternately in 16h of darkness and at the temperature of 28 ℃ and the rotation speed of 160rpm for 7 days.

Further, in the step (3), the Sevage reagent is prepared by mixing the following components in a volume ratio of 4:1, trichloromethane-isopropanol mixed solution; the dosage of the Sevage reagent is equal to the volume of the sample, and the cut-off molecular weight of a dialysis bag adopted for dialysis is 3500 Da.

Further, in the step (4), an anion exchange chromatography column is DEAE Sepharose Fast Flow, the size is 2.0cm × 30cm, the gradient eluent is 300mL Tris-HCl buffer solutions containing NaCl with different concentrations, the pH value is 7.4, the NaCl concentration gradient is 0, 0.3mol/L and 0.5mol/L, and the collected elution peak is a component eluted by 0.5mol/L NaCl.

Further, in the step (5), the size exclusion chromatography is performed by using Sephadex G-75 with a well-balanced 0.01mol/L NaCl solution, the size is 1.0cm × 50cm, and the eluent is 0.01mol/L NaCl solution.

The chlorella extracellular polysaccharide compound is applied to inhibiting inflammation.

Experimental results show that the chlorella extracellular polysaccharide has an obvious inflammation inhibiting effect, and can obviously reduce the generation of inflammatory factors ROS, iNOS, IL-6 and TNF- α.

The invention has the beneficial effects that:

(1) the chlorella extracellular polysaccharide compound has immunoregulation activity, is extracted from chlorella supernatant and is a natural extract, and has good safety;

(2) the chlorella extracellular polysaccharide compound CPEPS-2 obtained by the invention is a pure product;

(3) the chlorella extracellular polysaccharide compound obtained by the invention has good inflammation inhibiting effect;

(4) the chlorella extracellular polysaccharide compound obtained by the invention is simple to prepare, and can be combined with intracellular grease extraction, so that the cost of the microalgae grease is further reduced.

Drawings

FIG. 1 is an ion exchange chromatogram of DEAE Sepharose Fast Flow of the Chlorella vulgaris exopolysaccharide complex of the present invention.

FIG. 2 is a Sephadex G-75 gel column analysis diagram of the chlorella exopolysaccharide complex of the present invention.

FIG. 3 is a high performance liquid chromatography of the chlorella exopolysaccharide complex of the present invention.

FIG. 4 is a Fourier infrared spectrum of the Chlorella exopolysaccharide complex of the present invention.

FIG. 5 is the nuclear magnetic resonance hydrogen spectrum of the chlorella exopolysaccharide complex of the present invention.

FIG. 6 is a graph showing the effect of the complex of extracellular polysaccharides of Chlorella vulgaris of the present invention on the production of ROS by macrophage RAW264.7 under stimulation of LPS, wherein the graph indicates a significant difference (P < 0.05) compared to the blank control group and indicates a very significant difference (P < 0.01); a represents a significant difference (P < 0.05) compared with the LPS group, and B represents a very significant difference (P < 0.01) (the same below).

FIG. 7 shows the effect of the complex of extracellular polysaccharides of Chlorella vulgaris of the present invention on iNOS in macrophage RAW264.7 under stimulation of LPS.

FIG. 8 shows the effect of the complex of extracellular polysaccharides of Chlorella vulgaris of the present invention on the production of IL-6 by the macrophage RAW264.7 under LPS stimulation.

FIG. 9 shows the effect of the complex of extracellular polysaccharides of Chlorella vulgaris of the present invention on the production of TNF- α by macrophage RAW264.7 under LPS stimulation.

Detailed Description

The present invention will be further described with reference to specific examples, but the present invention is not limited thereto.