WO2019223081A1 - 一种基于纤维素化磁性纳米颗粒富集和分离厌氧纤维降解菌的方法 - Google Patents

一种基于纤维素化磁性纳米颗粒富集和分离厌氧纤维降解菌的方法 Download PDF

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WO2019223081A1
WO2019223081A1 PCT/CN2018/095478 CN2018095478W WO2019223081A1 WO 2019223081 A1 WO2019223081 A1 WO 2019223081A1 CN 2018095478 W CN2018095478 W CN 2018095478W WO 2019223081 A1 WO2019223081 A1 WO 2019223081A1
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magnetic nanoparticles
minutes
celluloseized
anaerobic
bacteria
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French (fr)
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赵圣国
王加启
郑楠
邢磊
张养东
李松励
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中国农业科学院北京畜牧兽医研究所
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Priority to US17/058,991 priority Critical patent/US20210214674A1/en
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    • B—PERFORMING OPERATIONS; TRANSPORTING
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    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/02—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material
    • B01J20/06—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material comprising oxides or hydroxides of metals not provided for in group B01J20/04
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    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
    • B01J20/24—Naturally occurring macromolecular compounds, e.g. humic acids or their derivatives
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    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
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    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
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    • 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
    • C12N1/02—Separating microorganisms from their culture media
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    • 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
    • C12N1/20—Bacteria; Culture media therefor
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    • 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
    • C12N1/22—Processes using, or culture media containing, cellulose or hydrolysates thereof
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    • C12N13/00—Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves

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  • the invention belongs to the field of biotechnology, and particularly relates to a method for enriching and isolating anaerobic fiber degrading bacteria based on celluloseized magnetic nanoparticles.
  • Cellulose is an essential nutrient for humans or animals, and degradation of cellulose is accomplished by anaerobic fiber-degrading bacteria in the gastrointestinal tract.
  • Previous studies have found that the diversity of anaerobic fiber-degrading bacteria in the gastrointestinal tract is high and more than 90% have not been isolated and cultured.
  • Common methods for microbial separation and culture include dilution culture and microbial co-culture. However, these methods have the disadvantages of low separation efficiency, complicated operation, expensive equipment or lack of group interaction, and it is difficult to isolate new specific functions of anaerobic fiber-degrading bacteria.
  • the primary object of the present invention is to provide a method for enriching and isolating anaerobic fiber degrading bacteria based on celluloseized magnetic nanoparticles;
  • Another object of the present invention is to provide a method for preparing celluloseized magnetic nanoparticles used in the method.
  • Another object of the present invention is to provide a celluloseized magnetic nanoparticle prepared by the above preparation method.
  • Another object of the present invention is to provide the use of the above-mentioned celluloseized magnetic nanoparticles, which can be used for enrichment of anaerobic fiber-degrading bacteria and isolation of anaerobic fiber-degrading bacteria.
  • a method for enriching and isolating anaerobic fiber degrading bacteria based on celluloseized magnetic nanoparticles includes the following steps:
  • bacteria Adding bacteria to the solution containing celluloseized magnetic nanoparticles, the bacteria containing anaerobic fiber degrading bacteria; mixing and incubating; and then using magnet adsorption for separation to obtain anaerobic fiber degrading bacteria;
  • the celluloseized magnetic nanoparticles include magnetic nanoparticles and a cellulose layer coated on the surface of the particles.
  • the magnetic nanoparticles are selected from the group consisting of ferric oxide nanoparticles.
  • the average particle diameter of the magnetic nanoparticles is 10-30 nm, such as 18 nm, 20 nm, and 22 nm.
  • the solution containing celluloseized magnetic nanoparticles is prepared by adding celluloseized magnetic nanoparticles to an anaerobic diluent.
  • the concentration of the celluloseized magnetic nanoparticles is 0.1-10 mg / mL, such as 8.70 mg / mL, 5.80 mg / mL, 4.35 mg / mL, 1.74 mg / mL, 0.87 mg / mL, 0.44 mg / mL.
  • the mL is preferably 4.35-10 mg / mL, and more preferably 8.70 mg / mL.
  • the concentration of the bacteria is (0.8-1.2) ⁇ 10 8 CFU / mL, preferably 1.0 ⁇ 10 8 CFU / mL.
  • the mixing time may be 5 minutes to 60 hours, preferably 5 to 15 minutes, such as 5 minutes, 8 minutes, 9 minutes, 10 minutes, and 15 minutes.
  • the incubation time does not exceed 60 hours, and may be 0-60 hours, preferably 12-60 hours, such as 12 hours, 24 hours, 48 hours, and 60 hours.
  • anaerobic fiber-degrading bacteria decomposing cellulose on the surface of celluloseized magnetic nanoparticles, and release of anaerobic fiber-degrading bacteria are achieved. Because cellulose has a high-efficiency affinity for bacterial cell walls, bacteria can adhere to the surface of celluloseized magnetic nanoparticles. Anaerobic fiber-degrading bacteria have a decomposition effect on cellulose, which can gradually decompose cellulose on the surface of celluloseized magnetic nanoparticles, and the anaerobic fiber-degrading bacteria are freed in a liquid environment; the liquid is, for example, selected from an anaerobic dilution solution.
  • the use of magnet adsorption for separation can be specifically: using magnet adsorption, then sucking the supernatant and adding the anaerobic diluent, and then adding the anaerobic diluent after washing.
  • the number of times of cleaning can be 1-3 times, such as 1 time, 2 times, and 3 times.
  • the present invention also provides a method for preparing celluloseized magnetic nanoparticles used in the above method, including:
  • the magnetic nanoparticles and the cellulose dispersion are mixed and reacted to obtain celluloseized magnetic nanoparticles.
  • the celluloseized magnetic nanoparticles include magnetic nanoparticles and a cellulose layer coated on the surface of the particles.
  • the concentration of the magnetic nanoparticles is 7-28 mg / mL, preferably 10-25 mg / mL, for example, about 10, 11, 12, 13, 14, 15 , 16, 17, 18, 19, or 20 mg / mL.
  • the average particle diameter of the magnetic nanoparticles is 10-30 nm, such as 18 nm, 20 nm, and 22 nm.
  • the method for preparing the magnetic nanoparticles includes, but is not limited to, performing a ultrasonic reaction on a mixture of FeCl 2 and FeCl 3 under alkaline conditions to obtain magnetic nanoparticles.
  • the base used in the alkaline condition may be NaOH.
  • the alkaline solution in the cellulose dispersion may be sodium hydroxide, urea, or a mixed solution of the two.
  • the temperature of the reaction may be room temperature.
  • the reaction time may be 5-15 minutes, such as 5 minutes, 8 minutes, 9 minutes, 10 minutes, and 15 minutes.
  • the preparation method further includes the steps of magnetically separating and washing the celluloseized magnetic nanoparticles after the reaction is completed.
  • the magnet capture time in the magnetic separation step may be 2-8 minutes, such as 3 minutes, 5 minutes, and 7 minutes.
  • the applicant uses DNS method to evaluate whether the surface of magnetic nanoparticles is coated with cellulose, as shown in FIG. 3.
  • Figure 3 shows that the surface of the magnetic nanoparticles was successfully coated with cellulose.
  • the present invention also provides a celluloseized magnetic nanoparticle prepared by using the above preparation method, which includes magnetic nanoparticle and a cellulose layer coated on the surface of the particle.
  • the invention also provides the use of the above-mentioned celluloseized magnetic nanoparticles, which can be used to enrich and isolate anaerobic fiber-degrading bacteria.
  • the mechanism of the present invention is shown in Figure 1.
  • the celluloseized magnetic nanoparticles of the present invention include magnetic nanoparticles and a cellulose layer coated on the surface of the particles, wherein the cellulose has a high-efficiency affinity for the bacterial cell wall.
  • Bacteria can be directionally adhered; after the adhesion is completed, cellulose is decomposed by the anaerobic fiber-degrading bacteria to gradually decompose cellulose on the surface of the celluloseized magnetic nanoparticles until the anaerobic fiber-degrading bacteria and cellulose
  • the magnetic nanoparticles lose their connection and are then free from the liquid environment.
  • Non-anaerobic fiber-degrading bacteria still adhere to the surface of the corresponding celluloseized magnetic nanoparticles.
  • Magnetic nanoparticles have superparamagnetic properties and will aggregate under the action of an external magnetic field. In order to achieve the separation and enrichment of anaerobic fiber-degrading bacteria and non-anaerobic fiber-degrading
  • the present invention has the following advantages and effects:
  • the magnetic enrichment method is faster than the culture medium enrichment method, the separation efficiency is high, the operation is more convenient, the price of the equipment used is low, and a lot of time and human, material and financial resources are saved.
  • the capture efficiency of bacteria is stronger.
  • the bacterial binding efficiency of the fibrotic magnetic nanoparticles reaches more than 90%, or even more than 95%.
  • the bacterial concentration is 1.0 ⁇ 10 8 CFU / mL and the celluloseized magnetic nanoparticle concentration is 8.7mg / mL, the bacterial binding efficiency Reached 99%.
  • the stability reached more than 80%, even more than 85%, and the best was 90.24%.
  • the invention can realize the enrichment of a large number of bacteria at the same time, instead of being limited to only certain bacteria.
  • FIG. 1 is a schematic diagram of a method for enriching and separating anaerobic fiber-degrading bacteria based on celluloseized magnetic nanoparticles.
  • FIG. 2 is a transmission electron microscope image (A) of a magnetic nanoparticle, a particle size statistics (B), a hysteresis curve (C), and a characterization of an X-ray diffraction pattern (D).
  • Fig. 3 is a graph showing the results of quantitative evaluation of cellulose on the surface of celluloseized magnetic nanoparticles.
  • Fig. 4 is a graph showing the binding ratio and efficiency evaluation of celluloseized magnetic nanoparticles and bacteria.
  • FIG. 5 is a diagram showing the binding stability of celluloseized magnetic nanoparticles and bacteria.
  • FIG. 6 is an evaluation diagram of binding specificity of celluloseized magnetic nanoparticles and anaerobic fiber degrading bacteria.
  • the formula of the medium is: 1g glucose, 1g peptone, 6.0g / L K 2 HPO 4 , 1.21g / L CaCl 2 , 6.0g / L KH 2 PO 4 , 12.0g / L NaCl, 6.0g / L (NH 4 ) 2 SO 4 , 12.5 g / L MgSO 4 ⁇ 7H 2 O, 0.5 mL heme; prepare the components according to the formula and mix, and then continuously pass in CO 2 for 4 hours after boiling, adjust the pH to 6.8, After adding 0.125g of cysteine hydrochloride, cover quickly. Place the configured medium in an anaerobic glove box, aliquot it into an anaerobic culture tube, autoclave at 121 MPa for 15 minutes, and prepare the anaerobic medium for later use.
  • the formula of the diluent is: 6.0g / L K 2 HPO 4 , 1.21g / L CaCl 2 , 6.0g / L KH 2 PO 4 , 12.0g / L NaCl, 6.0g / L (NH 4 ) 2 SO 4 , 12.5g / L MgSO 4 ⁇ 7H 2 O, 0.5mL hemoglobin; prepare and mix the components according to the formula, continuously pass in CO 2 for 4 hours after boiling, adjust the pH to 6.8, and add 0.125g cysteamine Cover quickly after the hydrochloride. Place the configured medium in an anaerobic glove box, aliquot it into an anaerobic culture tube, autoclave at 121 MPa for 15 minutes, and prepare the anaerobic diluent for later use.
  • Streptococcus bovis non-fiber degrading bacteria
  • Cellulomonas fiber degrading bacteria
  • Streptococcus bovis and Cellulomonas are strictly anaerobic bacteria. Streptococcus bovis and Cellulomonas stored in the laboratory were respectively inoculated into anaerobic culture tubes containing anaerobic medium, and cultured at 37 ° C for 48h.
  • the main purpose of this example is to evaluate the cellulose coating on the surface of the celluloseized magnetic nanoparticles.
  • the celluloseized magnetic nanoparticles were dissolved in an anaerobic dilution solution to prepare a solution of celluloseized magnetic nanoparticles at a concentration of 17.4 mg / ml. 3 ml of the above-mentioned solution of celluloseized magnetic nanoparticles, water (negative control) and Hydroxymethylcellulose sodium (positive control, 5mg / ml), add 1ml cellulase solution (1mg / ml), 50 °C water bath for 30min, boiling water bath for 10min, after cooling to room temperature, add 3ml DNS solution, boiling water bath for 10min After cooling to room temperature, make up to 25ml, and use a spectrophotometer to detect the OD value at a wavelength of 550nm. The results are shown in Fig. 3. It can be seen from Fig. 3 that after the cellulase treatment, the celluloseized magnetic nanoparticles released reducing sugar, indicating that the surface of the magnetic nanoparticles was successfully coated with cellulose.
  • the main purpose of this embodiment is to obtain the optimal concentration of the celluloseized magnetic nanoparticles and bacteria, and the capture efficiency of bacteria.
  • the celluloseized magnetic nanoparticles were dissolved in an anaerobic dilution solution to prepare a solution, and the celluloseized magnetic nanoparticles were diluted to 8.70, 5.80, 4.35, 1.74, 0.87, 0.44 mg / mL and Streptococcus bovis (1.0 ⁇ 10, respectively).
  • the main purpose of this example is to evaluate the long-term stability of the binding of celluloseized magnetic nanoparticles to bacteria.
  • the celluloseized magnetic nanoparticles were dissolved in an anaerobic dilution solution to prepare a solution of the celluloseized magnetic nanoparticles at a concentration of 8.70 mg / mL.
  • a solution of 0.5 mL of the above celluloseized magnetic nanoparticles and 0.5 mL of Streptococcus bovis were prepared. (1.0 ⁇ 10 8 CFU / mL), and after mixing for 10 minutes, the cellulose-based magnetic nanoparticles are adsorbed by a magnet, and then the supernatant is added and an equal volume of anaerobic diluent is added. After washing twice, the anaerobic diluent is added.
  • the main purpose of this example is to evaluate the specificity of the separation of celluloseized magnetic nanoparticles.
  • Dissolve celluloseized magnetic nanoparticles in anaerobic diluent prepare a solution, mix equal volume of Streptococcus bovis and Cellulomonas, take 0.5mL and combine with 0.5mL celluloseized magnetic nanoparticles, and mix thoroughly after 10 minutes , Use a magnet to absorb cellulose-based magnetic nanoparticles, discard the liquid, replace it with an equal volume of anaerobic diluent, repeat the operation twice, incubate at 30 ° C, and extract the suspension at 0h, 12h, 24h, 36h, 48h, and 60h to extract DNA.
  • the above products were subjected to DNA extraction using a bacterial genomic DNA extraction kit (purchased from Tiangen Biochemical Technology Co., Ltd.).
  • the qPCR reaction program was: pre-denaturation at 95 ° C for 30s, 95 ° C for 5s, 60 ° C for 30s, 72 ° C for 34s, and 40 cycles.

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Abstract

一种基于纤维素化磁性纳米颗粒富集和分离厌氧纤维降解菌的方法。在磁性纳米表面包被纤维素制备纤维素化磁性纳米颗粒，细菌与纤维素化磁性纳米颗粒结合，厌氧纤维降解菌分解纤维素后脱离纤维素化磁性纳米颗粒，通过磁铁分离获得厌氧纤维降解菌，磁性纳米分离技术能够作为一种新的高效分离厌氧纤维降解菌的技术使用。富集和分离厌氧纤维降解菌的方法具有快速、分离效率高、操作方便、使用的设备价格低、对细菌的捕获效率强、对细菌的富集范围广，而不单单局限于某种细菌、灵敏度高、结果的准确性高等优点，具有工业化的应用前景。

Description

一种基于纤维素化磁性纳米颗粒富集和分离厌氧纤维降解菌的方法 技术领域
本发明属于生物技术领域，具体涉及一种基于纤维素化磁性纳米颗粒富集和分离厌氧纤维降解菌的方法。
背景技术
纤维素是人体或动物一种必需的营养素，而纤维素的降解是由胃肠道中厌氧纤维降解菌来完成。前期研究发现，胃肠道中厌氧纤维降解菌群多样性高且90％以上仍未被分离和培养。常见的微生物分离培养方法主要包括稀释培养法、微生物共培养法等。然而，这些方法存在分离效率低、操作复杂、设备昂贵或缺乏群体交互作用等缺点，难以分离新的特定功能的厌氧纤维降解菌。
发明内容
为了克服现有技术的缺点和不足，本发明的首要目的在于提供一种基于纤维素化磁性纳米颗粒富集和分离厌氧纤维降解菌的方法；
本发明的另一目的在于提供所述方法中使用的纤维素化磁性纳米颗粒的制备方法。
本发明的另一目的在于提供一种由上述制备方法制备得到的纤维素化磁性纳米颗粒。
本发明的另一目的在于提供上述纤维素化磁性纳米颗粒的用途，该纤维素化磁性纳米颗粒可用于厌氧纤维降解菌的富集以及厌氧纤维降解 菌的分离。
本发明的目的通过以下技术方案实现：
一种基于纤维素化磁性纳米颗粒富集和分离厌氧纤维降解菌的方法，所述方法包括如下步骤：
向含有纤维素化磁性纳米颗粒的溶液中加入细菌，所述细菌中含厌氧纤维降解菌；进行混合和孵育；然后利用磁铁吸附，进行分离，得到厌氧纤维降解菌；
其中，所述纤维素化磁性纳米颗粒包括磁性纳米颗粒和包被于颗粒表面的纤维素层。
根据本发明，所述磁性纳米颗粒选自四氧化三铁纳米颗粒。
根据本发明，所述磁性纳米颗粒的平均粒径为10-30nm，例如18nm、20nm、22nm。
根据本发明，所述含有纤维素化磁性纳米颗粒的溶液是将纤维素化磁性纳米颗粒加入厌氧稀释液中配制而成。
根据本发明，所述纤维素化磁性纳米颗粒的浓度为0.1-10mg/mL，例如8.70mg/mL、5.80mg/mL、4.35mg/mL、1.74mg/mL、0.87mg/mL、0.44mg/mL，优选为4.35-10mg/mL，进一步优选为8.70mg/mL。
根据本发明，所述细菌的浓度为(0.8-1.2)×10 8CFU/mL，优选为1.0×10 8CFU/mL。
根据本发明，所述混合的时间可以为5分钟-60小时，优选为5-15分钟，例如5分钟、8分钟、9分钟、10分钟、15分钟。
根据本发明，所述孵育的时间不超过60小时，可以为0-60小时，优选为12-60小时，例如12小时、24小时、48小时、60小时。
根据本发明，在所述混合和孵育过程中，实现了细菌的粘附、厌氧纤维降解菌分解纤维素化磁性纳米颗粒表面的纤维素以及厌氧纤维降解 菌的游离。由于纤维素对细菌细胞壁有高效亲和作用，因此纤维素化磁性纳米颗粒表面可以粘附细菌。厌氧纤维降解菌对纤维素有分解作用，可使纤维素化磁性纳米颗粒表面的纤维素逐渐分解，进而厌氧纤维降解菌游离于液体环境中；所述液体例如选自厌氧稀释液。
根据本发明，所述利用磁铁吸附，进行分离可以具体为：利用磁铁吸附，然后吸取上清并加入厌氧稀释液，清洗后再加入厌氧稀释液。所述清洗的次数可以为1-3次，例如1次、2次、3次。
本发明还提供一种上述方法中使用的纤维素化磁性纳米颗粒的制备方法，包括：
(1)将纤维素分散在碱性溶液中，得到纤维素分散液；
(2)将磁性纳米颗粒和上述纤维素分散液混合，进行反应，得到纤维素化磁性纳米颗粒，所述纤维素化磁性纳米颗粒包括磁性纳米颗粒和包被于颗粒表面的纤维素层。
根据本发明，磁性纳米颗粒和纤维素分散液的混合体系中，所述磁性纳米颗粒浓度为7-28mg/mL，优选10-25mg/mL，例如约10、11、12、13、14、15、16、17、18、19或20mg/mL。
根据本发明，所述磁性纳米颗粒的平均粒径为10-30nm，例如18nm、20nm、22nm。
根据本发明，所述磁性纳米颗粒的制备方法包括但不限于：将FeCl 2和FeCl 3的混合物在碱性条件下进行超声反应，得到磁性纳米颗粒。优选地，所述碱性条件使用的碱可以为NaOH。优选地，超声反应结束后，还包括磁分离和清洗的步骤；其中，所述磁分离步骤中磁铁捕获的时间可以为2-8分钟，例如3分钟、5分钟、7分钟；所述清洗使用的溶剂为水，例如超纯水；所述清洗优选洗至pH＝7。
根据本发明，所述纤维素分散液中的碱性溶液可以为氢氧化钠、尿素或二者的混合溶液。
根据本发明，步骤(2)中，所述反应的温度可以为室温。
根据本发明，步骤(2)中，所述反应的时间可以为5-15分钟，例如5分钟、8分钟、9分钟、10分钟、15分钟。
根据本发明，所述制备方法还包括在反应完成后，对纤维素化磁性纳米颗粒进行磁分离，清洗的步骤。其中，所述磁分离步骤中磁铁捕获的时间可以为2-8分钟，例如3分钟、5分钟、7分钟。所述清洗使用的溶剂为水，例如超纯水。所述清洗优选洗至pH＝7。
本发明中，申请人利用DNS方法对磁性纳米颗粒表面是否包被了纤维素进行了评价，见图3。图3表明磁性纳米颗粒表面成功包被纤维素。
本发明还提供使用上述制备方法制备得到的纤维素化磁性纳米颗粒，其包括磁性纳米颗粒和包被于颗粒表面的纤维素层。
本发明还提供上述纤维素化磁性纳米颗粒的用途，该纤维素化磁性纳米颗粒可用于富集和分离厌氧纤维降解菌。
本发明的机理如图1所示，具体是：本发明的纤维素化磁性纳米颗粒包括磁性纳米颗粒和包被于颗粒表面的纤维素层，其中的纤维素对细菌细胞壁有高效亲和作用，可以对细菌进行定向粘附；粘附完成后，利用厌氧纤维降解菌对纤维素的分解作用，使纤维素化磁性纳米颗粒表面的纤维素逐渐分解，直至厌氧纤维降解菌与纤维素化磁性纳米颗粒失去连接，进而游离于液体环境中，非厌氧纤维降解菌仍粘附在相应的纤维素化磁性纳米颗粒表面；磁性纳米颗粒具有超顺磁性质，在外加磁场的作用下会聚集，从而实现厌氧纤维降解菌和非厌氧纤维降解菌分离与富集。
本发明相对于现有技术，具有如下的优点及效果：
(1)用磁性富集的方法相对于培养基增菌的方法更加快速，分离效率高，操作也更为方便，使用的设备价格低，节省了大量时间和人力、物力、财力。
(2)用纤维素对磁性纳米颗粒进行表面修饰后，其对细菌的捕获效率更强。所述纤维化磁性纳米颗粒的细菌结合效率达到90％以上，甚至95％以上，当细菌浓度为1.0×10 8CFU/mL，纤维素化磁性纳米颗粒浓度为8.7mg/mL时，细菌结合效率达到99％。细菌在纤维素化磁性纳米颗粒上稳定粘附60h时，稳定性达到80％以上，甚至85％以上，最优高达90.24％。本发明同时能实现对大量细菌的富集，而不是单单局限于某种细菌。
(3)基于qPCR的核酸检测分析方法相对于传统的分析方法更加快速，灵敏度也更高，结果的准确性也得到提高。
附图说明
图1是基于纤维素化磁性纳米颗粒对厌氧纤维降解菌进行富集和分离的方法原理图。
图2是磁性纳米颗粒的透射电镜图(A)、透射电镜图下的粒径统计(B)、磁滞回曲线图(C)、X射线衍射图的表征(D)。
图3是纤维素化磁性纳米颗粒表面纤维素定量的评价结果图。
图4是纤维素化磁性纳米颗粒与细菌结合比例与效率评价图。
图5是纤维素化磁性纳米颗粒与细菌结合稳定性图。
图6是纤维素化磁性纳米颗粒与厌氧纤维降解菌结合特异性评价图。
具体实施方式
下文将结合具体实施例对本发明做更进一步的详细说明。应当理解，下列实施例仅为示例性地说明和解释本发明，而不应被解释为对本发明 保护范围的限制。凡基于本发明上述内容所实现的技术均涵盖在本发明旨在保护的范围内。
下述实验方法如无特别说明，均为常规方法，所使用的实验材料如无特别说明，均可轻易地从商业公司获取。
实施例1
厌氧培养基的配制。所述培养基的配方为：1g葡萄糖，1g蛋白胨，6.0g/L K 2HPO 4,1.21g/L CaCl 2,6.0g/L KH 2PO 4,12.0g/L NaCl,6.0g/L (NH 4) 2SO 4,12.5g/L MgSO 4·7H 2O,0.5mL血红素；按所述配方准备所述组分并混合，煮沸后连续通入CO 2 4小时，调整pH为6.8后，加入0.125g半胱氨酸盐酸盐后迅速盖盖。将配置好的培养基放入厌氧手套箱分装至厌氧培养管，高压灭菌121MPa，15分钟，制得所述厌氧培养基备用。
厌氧稀释液的配制。所述稀释液的配方为：6.0g/L K 2HPO 4,1.21g/L CaCl 2,6.0g/L KH 2PO 4,12.0g/L NaCl,6.0g/L (NH 4) 2SO 4,12.5g/L MgSO 4·7H 2O,0.5mL血红素；按所述配方准备所述组分并混合，煮沸后连续通入CO 2 4小时，调整pH为6.8后，加入0.125g半胱氨酸盐酸盐后迅速盖盖。将配置好的培养基放入厌氧手套箱分装至厌氧培养管，高压灭菌121MPa，15分钟，制得所述厌氧稀释液备用。
牛链球菌(非纤维降解菌)和纤维单胞菌(纤维降解菌)的培养。牛链球菌和纤维单胞菌均为严格厌氧细菌，将实验室保存的牛链球菌和纤维单胞菌分别接种到含厌氧培养基的厌氧培养管中，37℃厌氧培养48h。
实施例2
将0.4970g FeCl 2和2.7030g FeCl 3混合，然后逐滴加入25ml NaOH(1.0mol/L)，使用超声机超声反应30分钟，再利用永久性磁铁底部分 离。吸取上清并用等体积超纯水替换，重复数次直到pH＝7，得到磁性纳米颗粒备用。对磁性纳米颗粒分别进行透射电子显微镜，磁滞回曲线，X射线衍射表征，结果见图2。从图2可见，磁性纳米颗粒平均直径20nm，磁性为3.3-24.9emu/g。
将8g纤维素溶解在400ml的碱性溶液(14g NaOH，24g尿素溶于162g水中配得)中充分混匀并在4℃过夜保存，得到纤维素分散液备用。将磁性纳米颗粒与纤维素分散液轻摇混合10分钟，使用永久性磁铁捕获5分钟，再使用超纯水清洗直至pH＝7，得到所述纤维素化磁性纳米颗粒。
实施例3
该实施例主要目的是评价纤维素化磁性纳米颗粒表面的纤维素包被情况。
将纤维素化磁性纳米颗粒溶于厌氧稀释液中，配制浓度为17.4mg/ml的纤维素化磁性纳米颗粒的溶液，取3ml上述纤维素化磁性纳米颗粒的溶液、水(阴性对照)和羟甲基纤维素钠(阳性对照,5mg/ml)，分别加入1ml纤维素酶液(1mg/ml)，50℃水浴30min，沸水浴10min，冷却至室温后，加入3ml DNS溶液，沸水浴10min，冷却至室温后定容至25ml，利用分光光度计在550nm波长下检测OD值。结果见图3，从图3可见，经过纤维素酶处理后，纤维素化磁性纳米颗粒释放出了还原糖，表明磁性纳米颗粒表面成功包被纤维素。
实施例4
该实施例主要目的是获得纤维素化磁性纳米颗粒与细菌结合的最优浓度，以及对细菌的捕获效率。
将纤维素化磁性纳米颗粒溶于厌氧稀释液中，配制溶液，分别将纤维素化磁性纳米颗粒稀释至8.70、5.80、4.35、1.74、0.87、0.44mg/mL与牛链球菌(1.0×10 8CFU/mL)按照体积比为5：5(总体积为1mL)混合10分钟后，放置用磁铁吸附纤维素化磁性纳米颗粒，弃液体，利用细菌基因组DNA提取试剂盒(购自天根生化科技有限公司)提取纤维素化磁性纳米颗粒吸附的细菌DNA，对牛链球菌定量，计算细菌与纤维素化磁性纳米颗粒的结合效率。结果见图4，从图4可见，随着纤维素化磁性纳米颗粒浓度的增加，牛链球菌吸附量增加，纤维素化磁性纳米颗粒浓度应至少4.35mg/ml，当纤维素化磁性纳米颗粒浓度为8.7mg/mL时，与细菌结合效率达到99％。
实施例5
该实施例的主要目的是评价纤维素化磁性纳米颗粒与细菌结合的长期稳定性。
将纤维素化磁性纳米颗粒溶于厌氧稀释液中，配制浓度为8.70mg/mL的纤维素化磁性纳米颗粒的溶液，将0.5mL上述纤维素化磁性纳米颗粒的溶液与0.5mL牛链球菌(1.0×10 8CFU/mL)结合，充分混合10分钟后利用磁铁吸附纤维素化磁性纳米颗粒，然后吸取上清并加入等体积的厌氧稀释液，清洗2次后再加入厌氧稀释液，置于4℃进行孵育，分别在0h、12h、24h、36h、48h、60h通过磁铁吸附，取悬液提取DNA，对牛链球菌进行qPCR定量。结果见图5，从图5可见，随着孵育时间的增加，牛链球菌有从纤维素化磁性纳米颗粒表面脱落的趋势，孵育60h时牛链球菌从纤维素化磁性纳米颗粒上脱落了9.76％。
实施例6
该实施例的主要目的是评价纤维素化磁性纳米颗粒的分离的特异性。
将纤维素化磁性纳米颗粒溶于厌氧稀释液中，配制溶液，将牛链球菌与纤维单胞菌等体积混合后取0.5mL与0.5mL纤维素化磁性纳米颗粒结合，充分混合10分钟后，使用磁铁吸取纤维素化磁性纳米颗粒，弃液体，使用等体积厌氧稀释液替换，重复操作两次，30℃培养，分别在0h、12h、24h、36h、48h、60h取悬液提取DNA，对牛链球菌(非纤维降解菌)和纤维单胞菌(纤维降解菌)进行qPCR定量。结果见图6，从图6可见，随着孵育时间的增加，纤维单胞菌(纤维降解菌)逐渐从纤维素化磁性纳米颗粒表面脱落进入溶液，而牛链球菌(非纤维降解菌)基本未从纤维素化磁性纳米颗粒表面脱落进入溶液，特异性分离纤维降解菌的效率为99.95％。
将上述产物用细菌基因组DNA提取试剂盒(购自天根生化科技有限公司)进行DNA提取。
通过设计的引物对提取的细菌基因组进行qPCR定量。
Figure PCTCN2018095478-appb-000001
qPCR体系：
上游引物 1
下游引物 1
TB Green Premix Ex Taq II(Tli RNaseH Plus)(2×) 12.5
DNA模板 2
灭菌水 8.5
总共 25μL
qPCR反应程序为：95℃预变性30s，95℃ 5s，60℃ 30s，72℃ 34s，40个循环。
从上述实施例3-6可见，在包被化方面，磁性纳米颗粒表面成功包被纤维素；在结合比例方面，当细菌浓度为1.0×10 8CFU/mL时，纤维素化磁性纳米颗粒浓度应调整至8.7mg/mL，细菌结合效率达到99％；在稳定性方面，细菌在纤维素化磁性纳米颗粒上稳定粘附60h，稳定性达90.24％，所以孵育时间优选为不超过60h；在特异性方面，纤维素化磁性纳米颗粒特异性分离厌氧纤维降解菌的效率为99.95％。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。

Claims (10)

  1. 一种基于纤维素化磁性纳米颗粒富集和分离厌氧纤维降解菌的方法，其特征在于，所述方法包括如下步骤：
    向含有纤维素化磁性纳米颗粒的溶液中加入细菌，所述细菌中含厌氧纤维降解菌；进行混合和孵育；然后利用磁铁吸附，进行分离，得到厌氧纤维降解菌；其中，所述纤维素化磁性纳米颗粒包括磁性纳米颗粒和包被于颗粒表面的纤维素层。
  2. 根据权利要求1所述富集和分离厌氧纤维降解菌的方法，其特征在于，所述磁性纳米颗粒选自四氧化三铁纳米颗粒；
    优选地，所述磁性纳米颗粒的平均粒径为10-30nm，例如18nm、20nm、22nm。
  3. 根据权利要求1或2所述富集和分离厌氧纤维降解菌的方法，其特征在于，所述含有纤维素化磁性纳米颗粒的溶液是将纤维素化磁性纳米颗粒加入厌氧稀释液中配制而成；
    优选地，所述纤维素化磁性纳米颗粒的浓度为0.1-10mg/mL，例如8.70mg/mL、5.80mg/mL、4.35mg/mL、1.74mg/mL、0.87mg/mL、0.44mg/mL，优选为4.35-10mg/mL，进一步优选为8.7mg/mL；
    优选地，所述细菌的浓度为(0.8-1.2)×10 8CFU/mL，优选为1.0×10 8CFU/mL；
    优选地，所述混合的时间可以为5分钟-60小时，优选为5-15分钟，例如5分钟、8分钟、9分钟、10分钟、15分钟；
    优选地，所述孵育的时间不超过60小时，可以为0-60小时，优选为12-60小时，例如12小时、24小时、48小时、60小时；
    优选地，所述利用磁铁吸附，进行分离可以具体为：利用磁铁吸附，然后吸取上清并加入厌氧稀释液，清洗后再加入厌氧稀释液；所述清洗的次数可以为1-3次，例如1次、2次、3次。
  4. 权利要求1-3任一项所述方法中使用的纤维素化磁性纳米颗粒的制备方法，其特征在于，所述制备方法包括：
    (1)将纤维素分散在碱性溶液中，得到纤维素分散液；
    (2)将磁性纳米颗粒和上述纤维素分散液混合，进行反应，得到纤维素化磁性纳米颗粒，所述纤维素化磁性纳米颗粒包括磁性纳米颗粒和包被于颗粒表面的纤维素层。
  5. 根据权利要求4所述的制备方法，其特征在于，磁性纳米颗粒和纤维素分散液的混合体系中，所述磁性纳米颗粒浓度为7-28mg/mL，优选10-25mg/mL，例如约10、11、12、13、14、15、16、17、18、19或20mg/mL；
    优选地，所述磁性纳米颗粒的平均粒径为10-30nm，例如18nm、20nm、22nm。
  6. 根据权利要求4或5所述的制备方法，其特征在于，所述磁性纳米颗粒的制备方法包括但不限于：将FeCl 2和FeCl 3的混合物在碱性条件下进行超声反应，得到磁性纳米颗粒；
    优选地，所述碱性条件使用的碱可以为NaOH；
    优选地，超声反应结束后，还包括磁分离和清洗的步骤；其中，所述磁分离步骤中磁铁捕获的时间可以为2-8分钟，例如3分钟、5分钟、7分钟；所述清洗使用的溶剂为水，例如超纯水；所述清洗优选洗至pH＝7。
  7. 根据权利要求4-6任一项所述的制备方法，其特征在于，所述纤维素分散液中的碱性溶液可以为氢氧化钠、尿素或二者的混合溶液。
  8. 根据权利要求4-7任一项所述的制备方法，其特征在于，步骤(2)中，所述反应的温度可以为室温；
    优选地，步骤(2)中，所述反应的时间可以为5-15分钟，例如5分钟、8分钟、9分钟、10分钟、15分钟；
    优选地，所述制备方法还包括在反应完成后，对纤维素化磁性纳米颗粒进行磁分离，清洗的步骤；
    优选地，所述磁分离步骤中磁铁捕获的时间可以为2-8分钟，例如3分钟、5分钟、7分钟；
    所述清洗使用的溶剂为水，例如超纯水；所述清洗优选洗至pH＝7。
  9. 根据权利要求4-8任一项所述的制备方法制备得到的纤维素化磁性纳米颗粒，其包括磁性纳米颗粒和包被于颗粒表面的纤维素层。
  10. 权利要求9所述纤维素化磁性纳米颗粒的用途，其特征在于，所述纤维素化磁性纳米颗粒可用于富集和分离厌氧纤维降解菌。
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