WO2011140839A1 - 一种两阶段高效生产发菜细胞及其胞外多糖的方法 - Google Patents

一种两阶段高效生产发菜细胞及其胞外多糖的方法 Download PDF

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WO2011140839A1
WO2011140839A1 PCT/CN2011/070579 CN2011070579W WO2011140839A1 WO 2011140839 A1 WO2011140839 A1 WO 2011140839A1 CN 2011070579 W CN2011070579 W CN 2011070579W WO 2011140839 A1 WO2011140839 A1 WO 2011140839A1
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culture
cells
medium
cell
stage
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戴玉杰
贾士儒
谭之磊
杨洪江
张峰
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天津科技大学
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/12—Unicellular algae; Culture media therefor

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  • the invention belongs to the field of biotechnology and relates to the field of cultivating cells of vegetative cells, and particularly relates to a method for efficiently producing hairy cells and extracellular polysaccharides thereof in two stages.
  • Candida albicans commonly known as hairy vegetables
  • hairy vegetables is a filamentous aggregate of terrestrial cyanobacteria (cyanobacteria), mainly distributed in the desert-semi-desert regions of northwest China and north China.
  • cyanobacteria terrestrial cyanobacteria
  • the photosynthesis of hairy vegetables, the fixation of carbon dioxide, and the ability to fix nitrogen, can fix the molecular nitrogen in the atmosphere to bioavailable nitrogen compounds, which is the main source of nitrogen in desert soils and can be used for the growth of other soil microorganisms.
  • bioavailable nitrogen compounds which is the main source of nitrogen in desert soils and can be used for the growth of other soil microorganisms.
  • Providing carbon sources and nitrogen sources people call them “pioneer creatures” in the desert, playing an important role in the energy and material circulation of ecologically fragile zones.
  • hairy vegetables have been favored by consumers, especially those in Fujian, Guangdong, Hong Kong, Macao and Taiwan and Southeast Asia. Hairy vegetables have strong adaptability to harsh environments.
  • a large amount of gelatinous substances are secreted outside the cells, which are wrapped around the hair cells and algae filaments to form a colloidal sheath to protect the hair cells from drought and high temperature. Damage to ultraviolet radiation.
  • the main component of the gelatinous substance is polysaccharide, and the polysaccharide is a kind of acidic heteropolysaccharide.
  • the polysaccharide has antiviral activity against various viruses such as influenza virus, human cytomegalovirus and herpes simplex virus. .
  • the polysaccharide can block the adsorption of the virus on the host cell, prevent the virus from replicating in the host cell, and has direct antiviral activity.
  • Japan and other countries have carried out various researches on the physiological functions of polysaccharides, and carried out research and development work on drugs and health products.
  • Patent (02138828. 8) "A method for cultivating hair cells”, which provides a method for cultivating hair cells, which is characterized in that a pulp slurry is obtained from the original hairy pulp and is gradually expanded. In the hairy cell culture, It provides a new way for liquid culture of hairy cells, but this method only uses photosynthetic autotrophic culture of hairy algae rather than hairy cells, and does not involve the production of extracellular polysaccharides by using hairy cells.
  • the method further comprises culturing the hairy vegetable cells, extracting the polysaccharide from the culture solution or directly producing the extracellular polysaccharide as a functional food or a drug.
  • the method has a greater growth rate than the wild hairy vegetable, the method Changed the status quo of extracting only polysaccharides from the genus Brassica, but because of the method of photoautotrophic culture, the photo-attenuation was affected in the later stage of culture, and the final harvested hair cells and extracellular polysaccharides were obtained. The output is low.
  • Patent (200510122059. 9) Solid culture method for hairy cells
  • Patent (200510122059. 9) Solid culture method for hairy cells
  • C12N1/12PF5483 Amaranth cell ⁇ solid culture method (C12N1/12PF5482)"
  • the method is to inoculate a hairy cell culture obtained by liquid suspension culture in a solid medium, and culture the hairy cells under artificial or natural conditions. And carry out dry and wet rhythm culture, provide controllable suitable culture conditions, and achieve complete artificial culture or field expansion culture.
  • This method breaks through the traditional cultivation method and accelerates the production speed, but this method can not realize the artificial planting industry of hairy vegetables. Production.
  • Patent (200610013589. 4) "High-density culture method for hairy cells with stable high-yield hairy polysaccharides" provides a method for high-density culture of hairy cells, which adopts a mode of mixed nutrition and heterotrophic culture, which can be shorter A large amount of hairy cells and extracellular polysaccharides are obtained in a timely manner.
  • This method breaks through the traditional photosynthetic autotrophic culture mode of the growth of the hairy cells, reduces the dependence on light during the cultivation process, and increases the hairy vegetables by adding an organic carbon source. The growth rate of the cells shortens the culture cycle of the hairy cells and increases the yield of the polysaccharides.
  • the culture method adds glucose to the culture medium at the beginning of the culture, it is highly susceptible to infection, so that the culture process is in progress.
  • the requirements for aseptic production conditions are very strict, which greatly increases the production cost and the risk of production, and is not conducive to the industrial production of hairy cells and their extracellular polysaccharides.
  • the hair culture methods reported in the literature are all cultured in a single stage, including photoautotrophic, mixed nutrition and heterotrophic culture.
  • Photosynthetic autotrophic culture has dependence on light, slow cell growth, low cell density, and cell.
  • the shortcomings of low-alcohol yield, mixed nutrition and heterotrophic culture have the disadvantages of low utilization rate of raw materials, easy contamination of bacteria, high requirements for aseptic conditions, high risk, and high production cost.
  • the object of the present invention is to overcome the deficiencies of the prior art, and to provide a two-stage method for efficiently producing hairy cells and extracellular polysaccharides thereof, which adopts a two-stage culture mode combining photoautotrophic and mixed nutrient culture. It effectively increases the growth rate of the hair cells and the utilization rate of glucose in the medium, and reduces the chance of infection and production cost, and simplifies the production process.
  • the first stage the seed cell seed solution is inoculated into the culture vessel, the medium is modified BG11 medium, cultured for 8-10 days, and the second is carried out when the concentration of the vegetable cells reaches 0.5-0.7 g/L. Stage culture
  • Second stage Under the same culture conditions as in the first stage, add glucose to a concentration of 1-20 g/L to the above culture medium, culture for 2-8 days, centrifuge the hair cell culture medium, and collect the hair. For the vegetable cells, the extracellular polysaccharide in the culture solution is extracted from the supernatant.
  • composition and content of the modified BG11 medium are:
  • the culture method of the step (1) is:
  • the seed cell seed solution was inoculated into the culture vessel at a seeding rate of 5-10% / v.
  • the medium was modified BG11 medium, initial pH 9.0, temperature 20-30 ° C, rotation speed 80-120 r / Min or ventilation ratio is 0.06-1.0wm, the light intensity is 2500-35001ux, culture is 8-10 days, and the next stage culture is carried out when the hairy cell concentration reaches 0.5-0.7g/L.
  • step of preparing the seed cell seed solution before the step (1) is:
  • the preserved hairy seed cell species are introduced into the culture solution, and the culture is statically cultured at 20-30 ° C.
  • the medium is modified BG11 medium, initial pH 9.0, light intensity is 800-12001 ux, and culture ends for 10-15 days. After culturing, the culture solution is allowed to stand until the cells are completely sedimented, the supernatant is discarded, and the cells are resuspended in sterile water to prepare a seed cell seed solution.
  • the invention establishes a two-stage high-efficiency production method of hairy vegetables and extracellular polysaccharides, that is, the first stage is photoautotrophic culture, and the second stage is photosynthetic autotrophic culture and heterotrophic culture mixed culture, and the method breaks through
  • the traditional hair cell growth and polysaccharide accumulation only adopt a single-stage culture mode, which makes full use of the advantages of photoautotrophic and mixed nutrition, and improves the growth rate of the hairy cells and the utilization rate of glucose in the medium.
  • glucose is added to the culture system, so that the medium can timely supplement the carbon source and the energy source, and the vegetable cells enter the rapid growth period, and at the same time, the glucose is added in the middle and late stages.
  • the utilization rate of glucose reduces the chance of contamination by bacteria and reduces the requirement of aseptic production conditions for hairy cell culture, thus effectively It reduces production risk and production cost, simplifies the production process, and explores a new way for the industrial production of vegetable cells and extracellular polysaccharides.
  • the two-stage culture mode is compared with the single photoautotrophic culture method, and the dry weight of the hairy cells produced in the similar culture time is increased by 5-8 times, and the extracellular polysaccharide yield is improved by nearly 15 times. -31 times, the cell dry weight is 3.0 ⁇ 5.0g/L, and the polysaccharide yield is 1.0 ⁇ 2.3 g/L, which effectively shortens the culture period of the hairy cells and increases the yield of extracellular polysaccharide, and the single polyculture method Compared with effectively reducing the infection rate, the production cost is significantly reduced.
  • the modified BG11 medium is used in the culture process of the present invention, and Na 2 Si (V9H 2 0 is added to the original BG11 medium, and the addition of the substance makes the growth of the hair cells more rapid and effectively improves the hair growth. Yield of vegetable cells and extracellular polysaccharides.
  • the culture mechanism of the present invention is:
  • the first stage is to inoculate vigorously growing hair cells into modified BG11 medium for liquid suspension culture.
  • the modified BG11 medium does not contain organic carbon source, and the hairy cells only use C0 2 in the air as carbon source. By fixing C0 2 by photosynthesis, it provides a carbon source for cell growth and reproduction.
  • the photosynthesis is affected by light attenuation, and the photosynthesis efficiency of unit cells is decreased, which cannot be the growth of hairy cells.
  • the production of extracellular polysaccharides provides sufficient energy, the growth of cells and the production of extracellular polysaccharides are initially inhibited. At this time, it is necessary to transfer to the second stage of culture, at which the cell concentration reaches 0.5-0.7 g/L.
  • the second stage is that after photosynthesis is affected by light attenuation, glucose is added at a final concentration of 1-2 g/L to provide sufficient carbon source and energy for the cells.
  • the cells continue to rapidly divide and grow, and a large amount of extracellular polysaccharides are formed.
  • continue to culture for 2-8 days stop the culture, separate and collect the cells, extract the extracellular polysaccharide in the culture solution, and add glucose to promote the secretion of extracellular polysaccharide.
  • the yield of polysaccharide is 1.0 ⁇ 2.3 g/L, and the dry weight of the cells reaches 3.0 ⁇ 5.0g/L.
  • the hairy cell species used in the embodiments of the present invention are isolated and purified menu cells isolated and purified from wild hairy vegetables in the laboratory, and the method for cultivating the vegetable seed cell seed liquid by using the menu cell species is:
  • the preserved hairy cell species were inserted into a triangular flask (500 mL) containing 200 mL of the culture solution, and cultured at 25 ° C, the medium was modified BG11 medium, initial pH 9.0, light intensity of 1000 lux, cells From slow growth to rapid growth, the culture is finished after 15 days of culture, the culture solution is allowed to stand, and the cells are completely sedimented, and the supernatant is discarded. The cells were resuspended in 200 mL of sterile water to prepare a seed cell seed solution.
  • composition and content of the above modified BG11 medium are:
  • a two-stage method for efficiently producing hairy cells and extracellular polysaccharides thereof the steps are as follows:
  • (1) First-stage culture of hairy cells The seedlings of the hairy cells are inoculated into a triangular flask (500 mL) containing 200 mL of culture medium at a dose of 5-10% (v/v).
  • the medium is modified BG11.
  • the culture medium initial pH 9.0, was cultured on a shaker at 25 ° C, the rotation speed was 100 rpm, the light intensity was 30001 ux, and the culture was carried out for 10 days. When the concentration of the hairy cells reached 0.5-0.7 g/L, the next step was carried out;
  • the second stage culture of the hairy cells After the first stage of culture for 10 days, the final concentration of 5 g/L of glucose is added to the culture solution, and the culture is continued for 5 days, and 200 mL of the cell culture medium is centrifuged at 4000 r/min for 10 minutes. Collect the hairpin cells, wash them twice with sterile water to remove the residual medium, dry to constant weight at 80 °C, determine the yield of the hairy cells, and take the alcohol from the centrifuged supernatant of the cell culture medium. The polysaccharide was separated and extracted by the method of sedimentation, and the yield of the crude polysaccharide was determined.
  • the dry weight of the hairy cells obtained in this example was 3.974 g/L, and the yield of extracellular polysaccharide was 1120.1 mg/L.
  • the method for extracting the polysaccharide of the hairy food by the alcohol precipitation method is as follows: adding the supernatant of the hairy cell culture medium to the centrifuged supernatant of the vegetable cell culture solution, 4 times the volume of absolute ethanol, and standing at 4 ° C for 12 hours, centrifuged at 4000 r / min. The extracellular polysaccharide was collected for 15 min, freeze-dried to obtain a polysaccharide product, and weighed.
  • a two-stage method for efficiently producing hairy cells and extracellular polysaccharides thereof the steps are as follows:
  • (1) First-stage culture of hairy cells Take the seed cell seed solution and inoculate it into a triangular flask (500 mL) containing 200 mL of culture solution at a dose of 5-10% (v/v). The medium is improved.
  • BG11 medium initial pH 9.0, cultured on a 25 ° C shaker, rotating at 100 rpm, light intensity of 30001 ux, cultured for 10 days;
  • the second stage culture of the hairy cells After the first stage of culture for 10 days, the final concentration of 10 g/L of glucose was added to the culture solution, and the culture was continued for 5 days. 200 mL of the cell culture medium was centrifuged at 4000 r/min for 10 minutes. The hair cells were washed twice with sterile water to remove the residual medium, and dried at 80 ° C to constant weight to determine the yield of the hair cells. The cell culture medium was centrifuged, and the polysaccharide was separated and extracted by alcohol precipitation to determine the yield of the crude polysaccharide.
  • a two-stage method for efficiently producing hairy cells and extracellular polysaccharides thereof the steps are as follows:
  • (1) First-stage culture of hairy cells The seedlings of the hairy cell were inoculated with 5 ⁇ 10% (v/v) inoculum into a 3L photobioreactor containing 2.5L of culture medium, and the aeration was 0.8wm.
  • the medium was modified BG11 medium, initial pH 9.0, culture temperature was 25 ° C, rotation speed was 100 rpm, light intensity was 35001 ux, and cell yield reached 0.6 g/L after 8 days of culture.
  • the second stage culture of the hairy cells After 8 days of the first stage, the final concentration of 5g/L glucose was added to the culture solution, and the culture was continued for 7 days. 200 mL of the cell culture medium was centrifuged at 4000 r/min for 10 minutes. The vegetable cells were washed twice with sterile water to remove the residual medium, dried at 80 ° C to constant weight, and the yield of the cabbage cells was measured. The hairy cell culture medium was separated and extracted by alcohol precipitation to determine the yield of the crude polysaccharide.
  • the dry weight of the hairy cells obtained in this example was 5.040 g/L, and the yield of extracellular polysaccharide was 1520.1 mg/L.
  • the seed cell seed solution was inoculated into a triangular flask (500 mL) containing 200 mL of culture medium at a dose of 5-10% (v/v).
  • the medium was modified BG11 medium, initial pH 9.0, at 25 °.
  • the seed cell seed solution was inoculated into a triangular flask (500 mL) containing 200 mL of the culture solution at a dose of 5-10% (v/v).
  • the medium was added 0.5 to 10 g/L on the basis of the modified BG11 medium.
  • Glucose initial pH 9.0, shaken at 25 °C, rotating at 100 rpm, light intensity 30001 ux, cultured for 15 days.

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Description

一种两阶段髙效生产发菜细胞及其胞外多糖的方法 技术领域
本发明属于生物技术领域， 涉及薇藻细胞的培养领域， 具体涉及一种两阶段高效 生产发菜细胞及其胞外多糖的方法。
背景技术
发菜 (Mostoc flagellifarme ), 学名是发状念珠藻， 俗称发菜， 是一种丝状聚合体 陆生蓝细菌 （蓝藻）， 主要分布于我国西北和华北的荒漠-半荒漠地区。 发菜进行光合 作用， 固定二氧化碳， 同时具有固氮能力， 可将大气中的分子态氮固定为生物可利用 的氮素化合物， 是荒漠地区土壤中氮元素的主要来源， 能为其它土壤微生物的生长提 供碳源和氮源， 因此， 人们称其为荒漠中的 "先锋生物"， 在生态脆弱带的能量与物质 循环中起着重要作用。
长期以来， 发菜作为一种食用蓝细菌， 受到广大消费者尤其是闽粵、 港澳台及东 南亚地区消费者的青睐。 发菜对恶劣环境具有极强的适应性， 其生长过程中向细胞外 分泌大量的胶状物质， 包裹于发菜细胞及藻丝外， 形成胶质鞘， 保护发菜细胞不受干 旱、 高温、 紫外辐射的损伤。 胶状物质的主要成分为多糖， 发菜多糖是一类酸性杂多 糖， 初步研究表明， 发菜多糖对流感病毒、 人巨细胞病毒、 单纯疱疹病毒等多种具封 套的病毒均具有抗病毒活性。 发菜多糖能阻断病毒对寄主细胞的吸附， 阻止病毒在寄 主细胞内的复制， 具有直接的抗病毒活性。 日本等国对发菜多糖的生理功能进行了多 方面的研究， 开展了有关药物和保健品的研发工作。
由于发菜具有很高的经济和药用价值， 因此，发菜和发菜多糖的需求量不断上升， 巨大的市场需求导致过渡采收造成发菜资源濒临枯竭， 从保护生态的角度出发， 我国 政府从 2000年 7月起就已禁止了对发菜的采收和销售，导致作为一种具有良好应用前 景的生物活性物质发菜多糖因原料资源匮乏而难以得到大规模的开发和生产。
为了解决市场需求不足和保护生态环境这一两难问题， 人们对人工培养发菜进行 了深入的研究， 主要包括固态培养法和液体培养法， 在不消耗天然发菜资源、 不破坏 发菜生长地生态环境的情况下扩大发菜的生产， 能够基本满足市场需求。
根据检索， 发现有以下几篇专利文献与本发明相关， 分别是：
1、 专利 （02138828. 8 ) "—种培养发菜细胞的方法"， 该方法提供了一种发菜细 胞的培养方法， 其特点是从原种发菜勾浆获得勾浆液， 经逐步扩大用于发菜细胞培养， 为发菜细胞的液体培养提供了一个新的途径， 但是该方法只采用发菜藻丝体而非发菜 细胞进行光合自养培养， 并且没有涉及利用发菜细胞生产胞外多糖。
2、 专利 （CN12144690O "—种发菜细胞培养联产发菜多糖的方法"， 该方法从野 生发菜藻体中分离获得离体细胞， 将分离得到的发菜细胞作为种子进行长期保藏， 用 于发菜细胞进一步培养， 从培养液中提取发菜多糖或直接作为功能性食品、 药物的生 产胞外多糖方法。 该方法与野生发菜相比， 生长速度有了较大的提高， 该方法改变了 只能从发菜藻体中提取发菜多糖的现状， 但由于该方法采用光合自养培养方法， 在培 养的后期受到光衰减的影响， 使最终收获的发菜细胞量和胞外多糖的产量都较低。
3、 专利 （200510122059. 9 ) "发菜细胞的固态培养方法"， 并在日本申请两项专 利： "髪菜細胞 培養 髪菜多糖 抽出 'J " $ 4 ΐ行 ^ t ¾ 方法
( C12N1/12PF5483 ) 髪菜細胞 ^固体培養方法（C12N1/12PF5482) "， 该方法是将液体 悬浮培养获得的发菜细胞培养物接种于固态培养基中， 在人工或自然条件下培养发菜 细胞， 并进行干湿节律培养， 提供可控的适宜培养条件， 实现完全人工培养或野外扩 大培养， 该方法突破了传统培养方式， 加快了生产速度， 但该方法还不能实现发菜的 人工种植产业化生产。
4、 专利 （200610013589. 4 ) "稳定高产发菜多糖的发菜细胞高密度培养方法"提 供了一种发菜细胞高密度培养的方法， 采用混合营养和异养培养的模式， 能在较短的 时间内获得大量发菜细胞和胞外多糖， 该方法突破了传统的发菜细胞生长的光合自养 培养模式， 减少了培养过程中对光的依赖性， 通过添加有机碳源可以提高发菜细胞的 生长速度， 缩短了发菜细胞的培养周期， 同时可以提高发菜多糖的产量， 但由于该培 养方法在培养开始时就在培养基中添加了葡萄糖， 极易染菌， 使培养过程中对无菌生 产条件要求非常严格， 从而大大提高了生产成本和生产的风险性， 不利于发菜细胞及 其胞外多糖的工业化生产。
目前文献报道的发菜培养方法都是采用单一阶段方式培养， 包括光合自养、 混合 营养和异养培养， 光合自养培养法存在对光照的依赖性、 细胞生长速度慢、 细胞密度 低、 胞外多糖产量低等缺点， 混合营养和异养培养存在原料利用率低、 容易污染杂菌、 无菌条件要求高、 风险性高、 生产成本高等缺点。
发明内容
本发明的目的在于克服现有技术的不足之处， 提供一种两阶段高效生产发菜细胞 及其胞外多糖的方法，该方法采用光合自养和混合营养培养相结合的两阶段培养模式， 有效提高了发菜细胞的生长速度和培养基中葡萄糖的利用率， 同时降低染菌几率和生 产成本， 简化生产工艺。
本发明的目的是通过以下技术方案实现的：
一种两阶段高效生产发菜细胞及其胞外多糖的方法，
(1)第一阶段： 取发菜细胞种子液接种到培养容器中， 培养基是改良的 BG11培养基， 培养 8-10天， 当发菜细胞浓度达到 0.5~0.7g/L时进行第二阶段培养；
(2)第二阶段： 在第一阶段相同的培养条件下， 向上述培养液中加入终浓度为 l~20g/L 的葡萄糖， 培养 2-8天， 将发菜细胞培养液离心， 收集发菜细胞， 取离心上清液提取培养 液中的胞外多糖。
而且， 所述改良的 BG11培养基成分及含量为：
NaN03 1.5g/L , K2HP04 0.04g/L , MgS04'7¾0 0.075g/L , CaCl2-2H20 0.036g/L , Na2Si03-9H20 0.058g/L , 柠檬酸 0.60mg/L， FeS04'¾0 4.80mg/L , H3B04 2.86mg/L， MnCl2-4H20 1.81mg/L, ZnS04-7H20 0.20mg/L, EDTA-2Na lmg/L, Na2Mo04 0.39mg/L, CuS04-5H20 0.079mg/L, CoCl2 0.01mg/Lo
而且， 所述步骤 (1)的培养方法为：
取发菜细胞种子液以 5~10% /v)的接种量接种到培养容器中，培养基是改良的 BG11 培养基， 初始 pH9.0， 温度为 20-30°C， 转速 80-120r/min或通风比 0.06-1.0wm， 光照强度 为 2500-35001ux， 培养 8-10天， 当发菜细胞浓度达到 0.5~0.7g/L时进行下一阶段培养。
而且， 在步骤 (1)前还包括制备发菜细胞种子液的步骤是：
将保存的发菜种细胞种接入培养液，在 20-30°C静置培养，培养基是改良的 BG11培养 基， 初始 pH9.0， 光照强度为 800-12001ux， 培养 10-15d后结束培养， 静置培养液至细胞完 全沉底， 弃去上清液， 细胞用无菌水重新悬浮， 制成发菜细胞种子液。
本发明的优点和积极效果是：
1、 本发明建立了一种发菜及其胞外多糖的两阶段高效生产方法， 即第一阶段是光合 自养培养， 第二阶段是光合自养培养与异养培养混合培养， 本方法突破了传统的发菜细 胞生长和多糖积累只采用单一阶段的培养模式， 充分利用了光合自养与混合营养的优点， 提高了发菜细胞的生长速度和培养基中葡萄糖的利用率。
2、 本发明在第一阶段培养 8-10后， 向培养体系中添加葡萄糖， 使培养基能够及时补 充碳源和能源， 发菜细胞进如快速生长期， 同时由于中后期添加葡萄糖， 提高了葡萄糖的 利用率， 减少了杂菌的污染机会， 降低了发菜细胞培养对无菌生产条件的要求， 从而有效 降低了生产风险和生产成本， 简化生产工艺， 为发菜细胞及胞外多糖的工业化生产探索出 一条新的途径。
3、 本发明涉及的方法中采用两阶段培养模式与单一光合自养培养方法相比， 在相近 的培养时间内生产的发菜细胞的干重提高 5-8倍， 胞外多糖产量提高近 15-31倍， 细胞干 重达 3.0~5.0g/L， 多糖产量达 1.0~2.3 g/L， 在有效缩短了发菜细胞的培养周期同时提高了 胞外多糖的产量， 与单一混养培养方法相比有效降低染菌率， 显著降低生产成本。
4、 本发明培养过程中采用改良的 BG11培养基， 在原来 BG11培养基培养基中添 加了 Na2Si(V9H20， 添加该物质后， 使发菜细胞的生长更加快速， 有效提高了发菜细 胞和胞外多糖的产量。
具体实施方式
以下结合附图对本发明的实施例做进一步详述； 本实施例是描述性的， 不是限定 性的， 不能由此限定本发明的保护范围。
下面结合实施例， 对本发明进一步说明， 下述实施例是说明性的， 不是限定性的， 不 能以下述实施例来限定本发明的保护范围。
本发明的培养机理是：
第一阶段是将生长旺盛的发菜细胞接种到改良的 BG11培养基中进行液体悬浮培养， 其中改良的 BG11培养基中不添加有机碳源， 发菜细胞仅以空气中的 C02为碳源， 通过光 合作用固定 C02， 为细胞的生长繁殖提供碳源， 当细胞培养到 8~10d后， 其光合作用受到 光衰减的影响， 单位细胞的光合作用效率下降， 不能为发菜细胞的生长和胞外多糖生成提 供充足能源时， 因此细胞的生长和胞外多糖的生成开始受到抑制， 这时需转入培养的第二 阶段， 此时的细胞浓度达到 0.5~0.7g/L。
第二阶段是光合作用受到光衰减的影响后， 添加终浓度为 l~20g/L的葡萄糖， 为细胞 提供充足的碳源和能源， 细胞继续快速分裂生长， 并且开始生成大量的胞外多糖， 继续培 养 2~8天， 停止培养， 分离收集细胞， 提取培养液中的胞外多糖， 添加葡萄糖后促进胞外 多糖的大量分泌， 多糖产量达 1.0~2.3 g/L， 细胞干重达到 3.0~5.0g/L。
本发明实施例采用的发菜细胞种是本实验室从野生发菜中分离纯化并保存的离体发 菜单细胞种， 利用发菜单细胞种培养发菜细胞种子液的方法为：
将保存的发菜细胞种接入装有 200mL培养液的三角瓶 （500mL) 中， 在 25 °C静置培 养， 培养基是改良的 BG11培养基， 初始 pH9.0， 光照强度为 lOOOlux, 细胞从缓慢生长过 渡到快速生长状态， 培养 15d后结束培养， 静置培养液， 等细胞完全沉底， 将上清液弃去， 细胞再用 200mL无菌水重新悬浮， 制成发菜细胞种子液。
上述改良的 BG11培养基成分及含量为：
NaN03 1.5g/L , K2HP04 0.04g/L , MgS04-7H20 0.075g/L , CaCl2'2¾0 0.036g/L , Na2Si03-9H20 0.058g/L , 柠檬酸 0.60mg/L， FeS04'¾0 4.80mg/L , H3BO4 2.86mg/L， MnCl2-4H20 1.81mg/L, ZnS04-7H20 0.20mg/L, EDTA-2Na lmg/L, Na2Mo04 0.39mg/L, CuS04-5H20 0.079mg/L, CoCl2 0.01mg/Lo
实施例 1 :
一种两阶段高效生产发菜细胞及其胞外多糖的方法， 步骤如下：
(1)发菜细胞第一阶段培养： 取发菜细胞种子液以 5~10% (v/v) 的接种量接种到装有 200mL培养液的三角瓶 （500mL) 中， 培养基是改良 BG11培养基， 初始 pH9.0， 在 25 °C 的摇床上培养， 转速 lOOrpm, 光照强度为 30001ux， 培养 10 天， 当发菜细胞浓度达到 0.5~0.7g/L时进行下一步；
(2)发菜细胞第二阶段培养： 当第一阶段培养 10天后， 向培养液中加入终浓度为 5g/L 的葡萄糖，继续培养 5天，取 200mL发菜细胞培养液 4000r/min离心 lOmin收集发菜细胞， 用无菌水洗涤两次， 以去除残留的培养基， 在 80°C烘干至恒重， 测定发菜细胞的产量， 取 发菜细胞培养液的离心上清液用醇沉法分离提取发菜多糖， 测定粗多糖的产量。
本实施例得到发菜细胞的干重是 3.974g/L， 胞外多糖产量是 1120.1 mg/L。
醇沉法提取发菜多糖的方法为： 向发菜细胞培养液离心上清中加入发菜细胞培养液离 心上清液 4倍体积的无水乙醇， 4°C静置 12h， 4000r/min离心 15min收集胞外多糖， 冷冻 干燥获得多糖产品， 称重。
实施例 2:
一种两阶段高效生产发菜细胞及其胞外多糖的方法， 步骤如下：
(1)发菜细胞第一阶段培养： 取发菜细胞种子液， 以 5~10% (v/v)的接种量接种到装有 200mL培养液的三角瓶（500mL)中， 培养基是改良的 BG11培养基， 初始 pH9.0， 在 25 °C 的摇床上培养， 转速 lOOrpm, 光照强度为 30001ux， 培养 10天；
(2)发菜细胞第二阶段培养： 当第一阶段培养 10天后， 向培养液中加入终浓度 10g/L的 葡萄糖， 继续培养 5天， 取 200mL发菜细胞培养液 4000r/min离心 lOmin收集发菜细胞， 用无菌水洗涤两次， 以去除残留的培养基， 在 80°C烘干至恒重， 测定发菜细胞的产量。 取 发菜细胞培养液离心上清液用醇沉法分离提取发菜多糖， 测定粗多糖的产量。
本实施例中得到细胞的干重是 4.910g/L， 胞外多糖产量是 2210.2 mg/L。 实施例 3 :
一种两阶段高效生产发菜细胞及其胞外多糖的方法， 步骤如下：
(1)发菜细胞第一阶段培养： 取发菜细胞种子液以 5~10% (v/v) 的接种量接种到装有 2.5L培养液的 3L光照生物反应器， 通气量为 0.8wm， 培养基是改良 BG11培养基， 初始 pH9.0， 培养温度为 25 °C， 转速 lOOrpm, 光照强度为 35001ux， 培养 8天后细胞产量达到 0.6g/L。
(2)发菜细胞第二阶段培养：当第一阶段 8天后，向培养液中加入终浓度 5g/L的葡萄糖， 继续培养 7天， 取 200mL发菜细胞培养液 4000r/min离心 lOmin收集发菜细胞， 用无菌水 洗涤两次， 以去除残留的培养基， 在 80°C烘干至恒重， 测定发菜细胞的产量。 取发菜细胞 培养液用醇沉法分离提取发菜多糖， 测定粗多糖的产量。
本实施例中得到发菜细胞的干重是 5.040g/L， 胞外多糖产量是 1520.1 mg/L。
产量对比试验： 发菜细胞经单一的光合自养培养获得细胞干重和多糖产量对比， 步骤 如下：
取发菜细胞种子液以 5~10% ( v/v ) 的接种量接种到装有 200mL 培养液的三角瓶 ( 500mL)中， 培养基是改良 BG11培养基， 初始 pH9.0， 在 25 °C摇床培养， 转速 100rpm， 光照强度为 30001ux， 培养 15天， 取 200mL发菜细胞培养液 4000r/min离心 lOmin收集发 菜细胞， 用无菌水洗涤两次， 以去除残留的培养基， 在 80°C烘干至恒重， 测定发菜细胞的 产量； 取发菜细胞培养液离心上清液用醇沉法分离提取发菜胞外多糖， 冷冻干燥后测定粗 多糖的产量， 据检测经单一培养得到发菜细胞的干重是 0.711g/L， 胞外多糖产量是 72.9mg/L。
染菌率对比： 发菜细胞经单一的混养培养的染菌率对比， 步骤如下：
取发菜细胞种子液以 5~10% ( v/v) 的接种量接种到装有 200mL培养液的三角瓶 ( 500mL)中，培养基是在改良 BG11培养基基础上添加 0.5~10g/L葡萄糖，初始 pH9.0， 在 25 °C摇床培养， 转速 100rpm， 光照强度为 30001ux， 培养 15天。 进行 10批次， 每 次 20瓶， 共 200瓶， 培养结束后共有 15瓶染菌， 染菌率 7.5%， 而采用实施例 1两阶 段法培养 10批次， 每次 20瓶， 培养结束后共染菌 3瓶， 染菌率 1.5%， 比混养培养染 菌率降低了 80%。

Claims

权利要求书
1、 一种两阶段高效生产发菜细胞及其胞外多糖的方法， 其特征在于：
(1)第一阶段： 取发菜细胞种子液接种到培养容器中， 培养基是改良的 BG11培养基， 培养 8-10天， 当发菜细胞浓度达到 0.5~0.7g/L时进行第二阶段培养；
(2)第二阶段： 在第一阶段相同的培养条件下， 向上述培养液中加入终浓度为 l~20g/L 的葡萄糖， 培养 2-8天， 将发菜细胞培养液离心， 收集发菜细胞， 取离心上清液提取培养 液中的胞外多糖。
2、 根据权利要求 1 所述的一种两阶段高效生产发菜细胞及其胞外多糖的方法， 其特 征在于： 所述改良的 BG11培养基成分及含量为：
NaN03 1.5g/L , K2HP04 0.04g/L , MgS04-7H20 0.075g/L , CaCl2'2¾0 0.036g/L ,
Na2Si03-9H20 0.058g/L , 柠檬酸 0.60mg/L， FeS04'¾0 4.80mg/L , H3B04 2.86mg/L , MnCl2-4H20 1.81mg/L, ZnS04-7H20 0.20mg/L, EDTA-2Na lmg/L, Na2Mo04 0.39mg/L, CuS04-5H20 0.079mg/L, CoCl2 0.01mg/Lo
3、 根据权利要求 1 所述的一种两阶段高效生产发菜细胞及其胞外多糖的方法， 其特 征在于： 所述步骤 (1)的培养方法为：
取发菜细胞种子液以 5~10% /v)的接种量接种到培养容器中，培养基是改良的 BG11 培养基， 初始 pH9.0， 温度为 20-30°C， 转速 80-120r/min或通风比 0.06-1.0wm， 光照强度 为 2500-35001ux， 培养 8-10天， 当发菜细胞浓度达到 0.5~0.7g/L时进行下一阶段培养。
4、 根据权利要求 1 所述的一种两阶段高效生产发菜细胞及其胞外多糖的方法： 其特 征在于： 在步骤 (1)前还包括制备发菜细胞种子液的步骤是：
将保存的发菜种细胞种接入培养液， 在 20-30°C静置培养， 培养基是改良的 BG11 培养基， 初始 pH9.0， 光照强度为 800-12001ux， 培养 10-15d后结束培养， 静置培养液 至细胞完全沉底， 弃去上清液， 细胞用无菌水重新悬浮， 制成发菜细胞种子液
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WO2013136029A1 (fr) 2012-03-16 2013-09-19 Fermentalg Production de polysaccharides en mode mixotrophe par nostoc
CN102669520A (zh) * 2012-05-07 2012-09-19 天津科技大学 一种发菜多糖口服液及其制备方法
CN114410708A (zh) * 2022-01-15 2022-04-29 陕西科技大学 一种提高发状念珠藻胞外多糖体外抗氧化活性和生物絮凝性的方法
CN114410708B (zh) * 2022-01-15 2023-09-29 陕西科技大学 一种提高发状念珠藻胞外多糖体外抗氧化活性和生物絮凝性的方法

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