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Fatty Acid Content and Composition of Triacylglycerols of Chlorella kessleri
凯氏小球藻中脂肪酸含量及三酰甘油组成   

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Scientific Reports
Jun 2016

Abstract

Triacylglycerols (TAGs) are esters formed from one glycerol and three fatty acids. TAGs are induced to accumulate in algal cells under environmental stress conditions including nutrient-limitation, hyperosmosis, and low temperature, for the storage of metabolic energy and carbon, and also for the consumption of excess energy (e.g., Hirai et al., 2016; Hayashi et al., 2017). Beside their physiological significance, the commercial utilization of algal TAG has been expected for the production of biodiesel, the methyl esters of fatty acids, from the aspect of carbon-neutral conception. The amounts of TAGs can be determined through quantitative measurement of their constituent fatty acids. This protocol consists of the following three parts: the first is the extraction of total lipids from algal cells with the use of organic solvents, chloroform and methanol, according to the method of Bligh and Dyer (1959), the second is the separation of TAG from the other lipid classes by thin-layer chromatography (TLC), and the third is the production of methyl-esterified derivatives of their constitutive fatty acids and subsequent quantitation of them by capillary gas-liquid chromatography (GLC). This protocol adapted from Sato and Tsuzuki (2011) is used for TAG analysis in a green alga, Chlorella kessleri.

Keywords: Chlorella kessleri (Chlorella kessleri), Gas-liquid chromatography (气液色谱法), Green algae (绿藻), Lipids (脂质), Thin-layer chromatography (薄层色谱法), Triacylglycerols (三酰甘油)

Background

Several methods have been used for determination of the fatty acid content of TAG. Simple and convenient protocols, e.g., include conversion of TAG to glycerol on treatment with a lipase, and subsequent measurement of the glycerol content through enzymatic generation of a product that reacts with a color- or fluorescence-generating probe (McGowan et al., 1983; Mendez et al., 1986). However, this enzymatic reaction based quantitation of TAG, inevitably, gives no information about the composition of constituent fatty acids. Meanwhile, HPLC provides information on TAG molecular species through their separation based on the numbers of carbon atoms and double bonds of constituent fatty acids, and enables their respective quantitation when combined with tandem mass spectrometer like in LC-MS/MS (Mu et al., 2000; Dorschel, 2002; MacDougall et al., 2011). The LC-MS/MS instrument, however, is very expensive. In this context, TLC/GLC based protocol for the measurement of the fatty acid content of TAG is introduced here, in view of the requirement of less expensive equipment than LC-MS/MS and definite information that can be obtained on quality and quantity of the constituent fatty acids.

Materials and Reagents

  1. 50 ml polypropylene centrifuge tubes with conical bottom (Corning, Falcon®, catalog number: 352070 )
  2. 50 ml glass screw cap centrifuge tubes with PTFE lined phenolic caps (AGC Techno Glass, catalog number: 8422CTF50 )
  3. 9 inch glass Pasteur pipettes (AGC Techno Glass, catalog number: IK-PAS-9P )
  4. TLC silica gel 60 glass plates 20 x 20 cm (Merck, catalog number: 105721 )
  5. Chromatography filter paper 1CHR 200 x 200 mm (GE Healthcare, catalog number: 3001-861 )
  6. Glass microcapillary pipette (Sigma-Aldrich, catalog number: Z543292 )
  7. 100 µl microsyringe (1710 RN, Hamilton, catalog number: 81030 ) with 22s/51/2 needle (Hamilton, catalog number: 7758-03 )
  8. 14 ml glass screw cap test tubes with PTFE lined phenolic caps (AGC Techno Glass, catalog number: TST-SCR16-125 )
  9. Inserts for large opening vials volume 0.15 ml (Sigma-Aldrich, catalog number: 24719 )
  10. 2 ml large opening vials with open-top screw cap (Sigma-Aldrich, catalog number: 29116-U )
  11. ULBON HR-Thermon-3000B GLC capillary column I.D. 0.25 x 25 m (Shinwa Chemical Industries)
  12. Chlorella kessleri 11 h, which corresponds to Parachlorella kessleri of NIES collection (http://mcc.nies.go.jp/) (National Institute for Environmental Studies, catalog number: NIES-2160 )
  13. Potassium chloride (KCl) (Wako Pure Chemical Industries, catalog number: 163-03545 )
  14. Butylated hydroxytoluene (BHT) (Wako Pure Chemical Industries, catalog number: 029-07392 )
  15. Methanol (CH3OH) (Wako Pure Chemical Industries, catalog number: 138-06473 )
  16. Chloroform (CHCl3) (Wako Pure Chemical Industries, catalog number: 033-08631 )
  17. Primuline (C21H15N3O3S3) (Tokyo Chemical Industry, catalog number: P0603 )
  18. Acetone (CH3COCH3) (Wako Pure Chemical Industries, catalog number: 016-00346 )
  19. Tripalmitin (C51H98O6) as TAG standard (Wako Pure Chemical Industries, catalog number: 200-03002 )
  20. N2 gas
  21. Hydrogen chloride-methanol reagent (5-10%) (Tokyo Chemical Industry, catalog number: X0041 )
  22. n-Hexane (C6H14) (Wako Pure Chemical Industries, catalog number: 084-03421 )
  23. Supelco 37 component FAME mix (Sigma-Aldrich, catalog number: 47885-U )
  24. Gamborg’s B5 medium salt mixture (Nihon Pharmaceutical, catalog number: 399-00621 ; Gamborg et al., 1968)
  25. Sorbitol (C6H14O6) (Wako Pure Chemical Industries, catalog number: 198-03755 )
  26. Arachidic acid (C20H40O2) (Tokyo Chemical Industry, catalog number: E0006 )
  27. 1/4 GB medium with or without 0.6 M sorbitol (see Recipes)
  28. Arachidic acid solution as an IS (see Recipes)

Equipment

  1. High-pressure steam sterilizer (TOMY SEIKO, model: LBS-245 )
  2. Tabletop centrifuge (Kubota, model: 5220 ) equipped with ST-720M swing rotor (16 x 50 ml)
  3. Vortex mixer (Scientific Industries, model: Vortex-Genie 2 )
  4. Rotary evaporator (Tokyo Rikakikai, model: N-1110V-W ) equipped with screw cap tube adaptor
  5. UV transilluminator (UVP, model: LM-20 )
  6. Fume hood (Yamato Scientific, model: KFS )
  7. Forced air flow oven (Tokyo Rikakikai, model: WFO-451SD )
  8. Spectrophotometer (Beckman Coulter, model: DU 640 )
  9. Double trough TLC chamber for 20 x 20 cm plates (Camag, catalog number: 022.5256 )
  10. Capillary gas-liquid chromatograph (Shimadzu, model: GC-2025 ) equipped with split/splitless injector, frame ion detector (FID) and autoinjector
  11. Chromatopac integrator (Shimadzu, model: C-R7A plus )
  12. Glass chromatographic reagent atomizer (Corning, PYREX®, catalog number: 2153-125 )

Procedure

  1. Total lipid extraction
    1. Culture C. kessleri cells at 30 °C in 100 ml of 1/4 GB (see Recipes) without sorbitol, under illumination (30 W m-2) and aeration for 48-72 h, to the late exponential phase of growth (the values of optical density at 730 nm [OD730] within ca. 0.4 to 0.6).
    2. Harvest cells through centrifugation in 2 x 50 ml tubes at 1,500 x g for 15 min at room temperature (tabletop centrifuge). Discard supernatant by decantation and resuspend cells in 20 ml of 1/4 GB with 0.6 M sorbitol for the induction of TAG accumulation (Hirai et al., 2016).
    3. Harvest cells through centrifugation at 1,500 x g for 15 min. Discard supernatant through decantation and resuspend cells in 20 ml of 1/4 GB with 0.6 M sorbitol. Repeat this centrifugation-resuspension step twice, finally with the OD730 value of cell culture adjusted to 0.3 in 50 ml of 1/4 GB with 0.6 M sorbitol. Culture cells for three days under the same growth conditions.
    4. Harvest cells through centrifugation at 1,500 x g for 15 min at room temperature. Discard supernatant through decantation and resuspend cells in 2 ml of 0.1 M KCl.
    5. Transfer cell suspension into a glass screw cap centrifuge tubes (50 ml vol.) with PTFE lined phenolic caps. Add 6.0 ml of methanol and 30 µl of 1.0% (w/v) BHT in methanol as an antioxidant agent to the sample, and agitate it by a vortex mixer for 30 sec at the maximum speed to destabilize cellular membrane systems.
    6. Add 3.0 ml of chloroform to the sample. Agitate it by a vortex mixer for 30 sec at the maximum speed and stand it for 10 min to extract lipids from the cells.
    7. Add 3.0 ml of chloroform once again and agitate the sample for intensive extraction of lipids by a vortex mixer for 30 sec at the maximum speed.
    8. Add 3.0 ml of distilled water and vortex the sample for 30 sec for emulsification. Centrifuge the mixture at 1,000 x g for 5 min at room temperature for its separation into three phases, i.e., the upper phase of H2O and methanol, the medium phase of cell debris, and the lower phase of chloroform. There is no cell debris at the bottom of the tube.
    9. Transfer the lower phase including total lipids with a Pasteur pipette into a glass screw cap centrifuge tubes (50 ml vol.) with PTFE lined phenolic caps. Be careful not to pick up the cell debris or the upper phase. The remaining lower phase can be recovered by the subsequent procedure.
    10. Add 3.0 ml of chloroform to the remaining upper and medium phases, and agitate the mixture for 30 sec with a vortex mixer. Centrifuge it at 1,000 x g for 15 min at room temperature.
    11. Recover the lower phase to the glass tube at Step A9.
    12. Repeat Steps A10 to A11 twice until lipids are fully recovered. Full recovery of lipids can be confirmed through observation of bleaching of the cell debris, which results from extraction of green chlorophylls into the lower phase.
      Note: Do not mechanically disrupt C. kessleri cells before lipid extraction, since the mechanical disruption would not increase the TAG recovery, but would decrease the TAG content with appearance of free fatty acids, owing probably to the action of endogenous lipases.
    13. Evaporate the solvent completely with a rotary evaporator, if necessary, with the addition of methanol or chloroform/methanol (2:1, v/v) for removal of residual H2O through azeotropy. Weigh dry residue and dissolve lipids in ca. 200 µl of chloroform/methanol (2:1, v/v), and transfer this total lipid fraction into a glass vial to be stored at -20 °C until use. The value of dry residue weight will be helpful for optimization of loading of total lipids on TLC plate for better separation of TAG.

  2. Separation of TAG from the other lipid classes by TLC
    1. Set a TLC chamber for the separation of TAG by placing a piece of filter paper along its wall, and then by pouring a developing solvent of hexane/diethylether/acetate (70:30:1, v/v) up to ca. 1 cm in depth. Close the lid for vapor saturation, which is promoted by the filter paper that has been soaked in the solvent. Stand the chamber for an hour or more to saturate it with the solvent vapor prior to the start of development. In order to ensure repeatability, the solvent is prepared freshly for each analysis.
    2. Heat a TLC silica gel 60 plate at 120 °C for a couple of hours in an oven to activate the silica gel just before development. Don’t touch the plate with bare hands during this procedure.
    3. Draw a horizontal line that is about 2 cm away from the bottom side of a TLC plate with a carbon pencil, and outline a horizontally long rectangle (ca. 2 x 3 cm) on the horizontal line of the plate. Lines have to be gently drawn to avoid scraping off silica gel. Don’t use an ink pen, otherwise, the lines would disappear.
      Note: Depending on the number of samples, the TLC plate may be cut into two pieces (10 cm wide ones) before use.
    4. Apply 45% of the stored total lipid fraction to the rectangle spot with a glass microcapillary pipette or 100 µl microsyringe with 22s/51/2 needle, and air-dry it. Repeat this step several times until this fraction is completely transferred to this spot.
    5. Apply 5-25 µl of standard TAG stock solution (1 mg/ml in chloroform, w/v) on the horizontal line next to the spot of the analyte samples.
    6. Place the TLC plate in the chamber, cover the chamber with its lid, and allow the plate to develop until the solvent goes up to about 2 cm below the top side of the plate.
    7. Remove the plate from the chamber and air-dry the plate.
    8. Spray the plate with primuline (0.01% in 80% acetone, w/v) in a fume hood, and illuminate it under 365 nm UV light to detect lipid compounds as exhibiting a pale color (see Figure 1). Outline the TAG spot with a pencil, judged from the position of TAG standard.
      Note: To prevent the TAG spot from diffusing, avoid spraying excessive detection reagent.
    9. Scrap off the silica gel of the TAG spot by a flat edge of micro-spatula. Silica gel from TAG should be immediately treated for the production of fatty acid methyl esters (FAMEs, see below) to prevent the oxidation of its constituent fatty acids.


      Figure 1. TLC separation of TAG in total lipid fraction in C. kessleri

  3. Conversion of constituent fatty acids into fatty acid methyl esters
    1. Take 50 µl of arachidic acid solution as an internal standard (IS, see Recipes) into glass screw cap test tubes (14 ml vol.) with PTFE lined phenolic cap, and dry it up under the flow of N2 gas.
      Note: Since Chlorella kessrelli 11 h has no endogenous C20 series fatty acids, the authors use arachidic acid (C20:0) as an IS. Nonadecanoic acid (C19:0) can also be used as an IS.
    2. Put scraped silica gel of TAG and 5% of the total lipid fraction into the test tubes, respectively. The latter lipid solution has to be air-dried under the flow of N2 gas. The remainder of the total lipid fraction, i.e., 50% of the initial level, can be stored at -80 °C for later analysis.
    3. Add 2.0 ml of 5-10% (w/v) hydrogen chloride-methanol reagent to each tube, cap it tightly, and agitate it by a vortex mixer for 30 sec at the maximum speed.
    4. Heat each tube at 95 °C for 2 h for methyl-esterification of the constituent fatty acids of TAG or total lipids.
    5. Cool down the tubes to room temperature, and add 2 ml of n-hexane to it. Agitate the tubes by a vortex mixer for 30 sec at a maximum speed for the extraction of FAMEs.
    6. Stand the tubes for 1 min for the separation of its content into upper (n-hexane) and lower (methanol) phases.
    7. Transfer the upper phase into a glass screw cap centrifuge tube (50 ml vol.) with PTFE lined phenolic caps by a glass Pasteur pipette.
    8. Add 2.0 ml of n-hexane to the remaining lower phase and vortex it for 30 sec. Stand the tube for 1 min at a room temperature.
    9. Recover the upper phase to the glass tube at Step C7.
    10. Repeat Steps C8 to C9 additionally two times for intensive extraction of FAMEs.
    11. Evaporate FAMEs solution with a rotary evaporator, and dissolve the dried samples in a small volume (< 50 µl) of n-hexane. Transfer the concentrated FAMEs solution into a 0.15 ml insert in a 2 ml glass vial, which is then capped for storage at 4 °C until measurement.
    12. Analyze fatty acid composition of the sample with a capillary GLC (see Figure 2). GLC is operated according to manufacturer’s instructions. GLC operating conditions are shown in Table 1.


      Figure 2. A gas chromatogram of FAMEs from TAG of C. kessleri

      Table 1. GLC operating conditions for determination of fatty acid composition

      Note: FID detects ions that are generated through burning of carbon compounds (Holm, 1999).

Data analysis

  1. Identify and calculate the peak areas of respective FAMEs on chromatogram with an integrator according to manufacturer’s instructions. FAMEs identification is based on the retention times as compared with those of the standard FAME mixture (Supelco 37 component FAME Mix, 100 µg/ml solution diluted with n-hexane). Check that chromatogram is being integrated properly with the appropriate baseline being shown.
  2. Quantitate each molecular species of FAME by using following Formula [1] with arachidic acid (20:0) as an IS. The values, 160 and 312, indicate the content of 20:0 (nmol) taken at Step C1 and the molecular weight of 20:0, respectively. The molar content of total lipids can be expressed on the basis of fatty acids that are included (Formula [2]).
  3. The TAG content is estimated as 1/3 of the summed molar contents of its constituent fatty acids (Formula [3]). Be careful that obtained values correspond to those included in respective samples that are analyzed. Alternatively, the content of TAG relative to that of total lipids is estimated on the basis of fatty acids (Formula [4]). As shown here, multiplication of ‘summed content of FAME from TAG’ by 1/9 is necessary in accordance with the quantitative ratio of the total lipid fraction used for TAG analysis (45%) to that for total lipid analysis (5%). The quantitative results should be presented by mean values ± standard deviations from at least three experiments.



    Note: The peak area of FAME is conveniently divided by the molecular weight of FA, but not by that of FAME, in view of full responses of FAME carbon atoms except its carbonyl one in FID (Ackman and Sipos, 1964).




Recipes

  1. 1/4 GB medium with or without 0.6 M sorbitol
    1. Dissolve Gamborg’s B5 medium salt mixture (3.3 g/packet, Table 2) in distilled water for preparation of normal Gamborg’s B5 medium (GB; final volume, 1 L)

      Table 2. Chemical composition of Gamborg’s B5 medium salt mixture


    2. Add 750 ml of distilled water to 250 ml of GB for preparation of 1/4 GB
    3. Otherwise, dissolve 109 g of sorbitol in 250 ml of GB with concomitant addition of distilled water for preparation of 0.6 M sorbitol containing 1/4 GB (final volume, 1 L)
    4. 1/4 GB and 0.6-M sorbitol containing 1/4 GB have to be subjected to autoclave sterilization (120 °C, 20 min)
  2. Arachidic acid solution as an IS
    1. Dissolve 10 mg of arachidonic acid in 10 ml 2-propanol
    2. Store the solution at 2-8 °C

Acknowledgments

This work was supported by Tokyo University of Pharmacy and Life Sciences. The authors have declared that no competing interests exist.

References

  1. , R.G. and Sipos, J. C. (1964). Application of specific response factors in the gas chromatographic analysis of methyl esters of fatty acids with flame ionization detectors. J Am Oil Chem Soc 41 (5): 377-378.
  2. Bligh, E. G. and Dyer, W. J. (1959). A rapid method of total lipid extraction and purification. Can J Biochem Physiol 37 (8): 911-917.
  3. Dorschel, C. A. (2002). Characterization of the TAG of peanut oil by electrospray LC-MS-MS. J American Oil Chemists’ Society 79 (8): 749-753.
  4. Gamborg, O. L., Miller, R. A. and Ojima, K. (1968). Nutrient requirements of suspension cultures of soybean root cells. Exp Cell Res 50 (1): 151-158.
  5. Hayashi, T., Otaki, R., Hirai, K., Tsuzuki, M. and Sato, N. (2017). Optimization of seawater-based triacylglycerol accumulation in a freshwater green alga, Chlorella kessleri, through simultaneous imposition of lowered-temperature and enhanced-light intensity. Algal Res 28: 100-107.
  6. Hirai, K., Hayashi, T., Hasegawa, Y., Sato, A., Tsuzuki, M. and Sato, N. (2016). Hyperosmosis and its combination with nutrient-limitation are novel environmental stressors for induction of triacylglycerol accumulation in cells of Chlorella kessleri. Sci Rep 6: 25825.
  7. Holm T. (1999). Aspects of the mechanism of the flame ionization detector. J Chromatogr A 842 (1-2): 221-227.
  8. MacDougall, K. M., McNichol, J., McGinn P. J., O’Leary, S. J. B. and Melanson, J. E. (2011). Triacylglycerol profiling of microalgae strains for biofuel feedstock by liquid chromatography-high-resolution mass spectrometry. Anal Bioanal Chem 401 (8): 2609-2616.
  9. McGowan, M. W., Artiss, J. D., Strandbergh, D. R. and Zak, B. (1983). A peroxidase-coupled method for the colorimetric determination of serum triglycerides. Clin Chem 29 (3): 538-542.
  10. Mendez, A. J., Cabeza, C. and Hsia, S. L. (1986). A fluorometric method for the determination of triglycerides in nanomolar quantities. Anal Biochem 156 (2): 386-389.
  11. Mu, H., Sillen, H. and Hiy, C. E. (2000). Identification of diacylglycerols and triacylglycerols in a structured lipid sample by atmospheric pressure chemical ionization liquid chromatography/mass spectrometry. J American Oil Chemists’ Society 77(10): 1049-1060.
  12. Sato, N. and Tsuzuki, M. (2011). Isolation and identification of chloroplast lipids. Methods Mol Biol 684: 95-104.

简介

三酰甘油(TAG)是由一种甘油和三种脂肪酸形成的酯。在包括营养限制,高渗和低温在内的环境胁迫条件下,TAG被诱导积累在藻细胞中,用于代谢能和碳的储存以及多余能量的消耗(例如, Hirai等人,2016; Hayashi等人,2017)。除了它们的生理意义之外,从碳中性概念的角度来看,藻类TAG的商业利用已经被期望用于生产生物柴油,即脂肪酸的甲酯。 TAG的量可以通过定量测定其构成脂肪酸来确定。该方案由以下三部分组成:首先是根据Bligh和Dyer(1959)的方法,使用有机溶剂,氯仿和甲醇从藻细胞中提取总脂质,其次是TAG的分离通过薄层色谱法(TLC)从其他脂质类别中分离出来,第三种是产生其组成型脂肪酸的甲基酯化衍生物,随后通过毛细管气相色谱法(GLC)对其进行定量。该协议改编自Sato和Tsuzuki(2011),用于绿藻的TAG分析, Chlorella kessleri 。

【背景】已经使用几种方法测定TAG的脂肪酸含量。简单而方便的方案包括用脂肪酶处理将TAG转化成甘油,随后通过酶产生与产生颜色或荧光的探针反应的产物测量甘油含量(McGowan等人,1983; Mendez等人,1986)。然而,这种基于酶反应的TAG定量不可避免地不提供关于构成脂肪酸组成的信息。同时,HPLC通过基于构成脂肪酸的碳原子数和双键的分离来提供关于TAG分子种类的信息,并且当与串联质谱仪(如在LC-MS / MS中一样)结合时使得它们各自定量, 2000年; Dorschel,2002; MacDougall等人,2011年)。然而,LC-MS / MS仪器非常昂贵。在这方面,基于TLC / GLC的TAG脂肪酸含量的测量方法介绍在这里,考虑到比LC-MS / MS更便宜的设备的要求和明确的信息,可以获得的质量和数量构成脂肪酸。

关键字Chlorella kessleri, 气液色谱法, 绿藻, 脂质, 薄层色谱法, 三酰甘油

材料和试剂

  1. 具有锥形底部的50ml聚丙烯离心管(Corning,Falcon ,目录号:352070)
  2. 50 ml带PTFE衬里酚醛盖的玻璃螺旋盖离心管(AGC Techno Glass,目录号:8422CTF50)
  3. 9英寸玻璃巴斯德移液器(AGC Techno Glass,目录号:IK-PAS-9P)
  4. TLC硅胶60块20×20厘米的玻璃板(Merck,目录号:105721)
  5. 色谱滤纸1CHR 200 x 200 mm(GE Healthcare,目录号:3001-861)
  6. 玻璃微毛细吸管(Sigma-Aldrich,目录号:Z543292)
  7. 具有22s / 51/2针(Hamilton,目录号:7758-03)的100μl微型注射器(1710 RN,Hamilton,目录号:81030)
  8. 14毫升带PTFE衬里酚醛盖的玻璃螺旋盖试管(AGC Techno Glass,目录号:TST-SCR16-125)

  9. 大开口小瓶的容量为0.15 ml(Sigma-Aldrich,目录号:24719)
  10. 2毫升带开口螺帽(Sigma-Aldrich,目录号:29116-U)的大开口小瓶
  11. ULBON HR-Thermon-3000B GLC毛细管柱I.D. 0.25×25米(信和化学工业)
  12. Chlorella kessleri 11 h,相当于NIES收集的 hlorense http://mcc.nies.go.jp/ )(国家环境研究所,目录号:NIES-2160)
  13. 氯化钾(KCl)(Wako Pure Chemical Industries,目录号:163-03545)
  14. 丁基羟基甲苯(BHT)(Wako Pure Chemical Industries,目录号:029-07392)
  15. 甲醇(CH 3 OH)(Wako Pure Chemical Industries,目录号:138-06473)
  16. 氯仿(CHCl3)(Wako Pure Chemical Industries,目录号:033-08631)
  17. Primuline(C 21 H 15 N 3 O 3 S 3)(东京,日本)化学工业,目录号:P0603)
  18. 丙酮(CH 3 = COCH 3)(Wako Pure Chemical Industries,目录号:016-00346)
  19. 作为TAG标准品(Wako Pure Chemical Industries,目录号:200-03002)的三棕榈酸甘油酯(C15H9N6O6)
  20. N 2气体
  21. 氯化氢 - 甲醇试剂(5-10%)(Tokyo Chemical Industry,目录号:X0041)
  22. -Hexane(C 6 H 14)(Wako Pure Chemical Industries,目录号:084-03421)。
  23. Supelco 37组分FAME混合物(Sigma-Aldrich,目录号:47885-U)
  24. Gamborg的B5中等盐混合物(Nihon Pharmaceutical,目录号:399-00621; Gamborg等人,1968)
  25. 山梨醇(C 6 H 14 O 6)(Wako Pure Chemical Industries,目录号:198-03755)
  26. 花生酸(C 20 H 40 O 2)(Tokyo Chemical Industry,目录号:E0006)
  27. 1 / 4GB中或不含0.6M山梨糖醇(见食谱)
  28. 花生酸溶液作为IS(见食谱)

设备

  1. 高压蒸汽灭菌器(TOMY SEIKO,型号:LBS-245)
  2. 配备ST-720M旋转转子(16×50毫升)的台式离心机(Kubota,型号:5220)
  3. 涡旋混合器(Scientific Industries,型号:Vortex-Genie 2)
  4. 旋转式蒸发器(东京Rikakikai,型号:N-1110V-W)配有螺旋盖管接头
  5. 紫外透射仪(UVP,型号:LM-20)
  6. 通风柜(大和科学,型号:KFS)
  7. 强制空气流动烤箱(东京Rikakikai,型号:WFO-451SD)
  8. 分光光度计(Beckman Coulter,型号:DU 640)
  9. 双槽TLC室20×20厘米板(Camag,目录号:022.5256)
  10. 装有分流/不分流进样器,框架离子检测器(FID)和自动注射器的毛细管气液色谱仪(岛津制作所,型号:GC-2025)
  11. Chromatopac积分仪(岛津,型号:C-R7A plus)
  12. 玻璃层析试剂雾化器(Corning,PYREX®,目录号:2153-125)

程序

  1. 总脂质提取
    1. 文化 C。 kessleri细胞在30℃下在100ml的1/4 GB(见配方)中没有山梨醇,在照射下(30Wm -2)和通气48-72小时至增长的指数后期(在730nm处的光密度值[OD 730℃]在0.4-0.6之间)。
    2. 通过在2×50ml试管中1500×g离心15分钟在室温(台式离心机)中收获细胞。通过滗析弃去上清液并将细胞重悬于20ml含0.6M山梨糖醇的1/4 GB的GB中以诱导TAG积累(Hirai等人,2016)。
    3. 通过在1500×g离心15分钟收获细胞。弃去上清液,用0.6M山梨糖醇将细胞重新悬浮在20毫升1 / 4GB的细胞中。重复该离心 - 再悬浮步骤两次,最后在0.6M山梨糖醇的50ml 1/4 GB的细胞培养物的OD 730值调节至0.3。在相同的生长条件下培养细胞三天。
    4. 通过在室温下1,500×g离心15分钟来收获细胞。通过倾析弃去上清液并将细胞重悬于2ml 0.1M KCl中。
    5. 将细胞悬浮液转移到带有PTFE衬里的酚醛盖的玻璃螺旋盖离心管(50毫升容积)中。向样品中加入6.0ml甲醇和30μl1.0%(w / v)BHT的甲醇溶液作为抗氧化剂,用涡旋混合器以最大速度搅拌30秒以使细胞膜系统不稳定。 >
    6. 向样品中加入3.0ml氯仿。用漩涡混合器以最大速度搅拌30秒,静置10分钟以从细胞中提取脂质。
    7. 再次加入3.0ml氯仿,搅拌样品,用涡旋混合器以最大速度强化萃取脂质30秒。
    8. 加入3.0ml蒸馏水并涡旋30秒以使样品乳化。在室温下,将混合物在1000×g下离心5分钟,使其分离成三相,即H 2 O的上相和甲醇,细胞碎片的中间相和氯仿的下层相。
      在管底部没有细胞碎片
    9. 用巴斯德移液管将包括总脂质的下层相转移到具有PTFE衬里的酚醛盖的玻璃螺旋盖离心管(50ml体积)中。小心不要拿起细胞碎片或上层相。剩下的下一阶段可以通过后续程序恢复。
    10. 向剩余的上,中相添加3.0ml氯仿,并用涡旋混合器搅拌混合物30秒。
      在室温下1000×g离心15分钟。

    11. 在步骤A9将下层相回收到玻璃管中。
    12. 重复步骤A10到A11两次,直到脂质完全恢复。通过观察细胞碎片的漂白来确认脂质的完全回收,这是由于将绿色叶绿素提取到下层相中所致。
      注意:由于机械破坏不会增加TAG回收率,但是由于内源性脂肪酶的作用,游离脂肪酸的出现可能会降低TAG含量,因此在脂质提取之前不要机械地破坏C. kessleri细胞。
    13. 如有必要,用旋转蒸发器完全蒸发溶剂,加入甲醇或氯仿/甲醇(2:1,v / v)以通过共沸除去残留的H 2 O.称取干燥的残余物并溶解200μl氯仿/甲醇(2:1,v / v)中的脂质并将该总脂质部分转移到玻璃小瓶中以在-20℃保存直到使用。干渣重量的值将有助于优化总脂质在TLC板上的加载以更好地分离TAG。

  2. 通过TLC从其他脂质类别中分离TAG
    1. 通过在其壁上放置一张滤纸,然后通过将展开剂己烷/乙醚/乙酸盐(70:30:1,v / v)倒入一个TLC室来分离TAG 。深度1厘米。关闭蒸汽饱和的盖子,这是由浸泡在溶剂中的滤纸促进的。在开发开始之前,将腔室放置一小时或更长时间以使溶剂蒸气饱和。为了确保可重复性,每次分析都要新鲜制备溶剂。
    2. 将TLC硅胶60板在烘箱中在120℃下加热几个小时以在显影之前激活硅胶。
      在这个过程中,请勿用手触摸板子
    3. 用碳素铅笔画一条距薄层板底部约2厘米的水平线,在水平线上画出一个水平长的矩形( ca。 2 x 3 cm)盘子。线必须轻轻拉伸,以避免刮擦硅胶。不要使用墨水笔,否则线条会消失。
      注意:根据样品的数量,TLC板可以在使用前切成两块(10厘米宽)。
    4. 用玻璃微毛细管吸取器或100μl微量注射器用22s / 51/2针将45%存储的总脂质部分施加到矩形斑点上,并风干。重复这个步骤几次,直到这个分数完全转移到这个地方。

    5. 在分析物样本点旁边的水平线上涂抹5-25μl标准TAG储备溶液(1 mg / ml氯仿,w / v)。
    6. 将TLC板置于室中,用盖子覆盖室,使板展开,直到溶剂上升到板上侧约2cm。

    7. 拆下培养皿中的培养皿,风干培养皿
    8. 在通风橱中用丙氨酸(0.01%,在80%丙酮中,w / v)喷洒板,并在365nm紫外光下照射以检测呈现浅色的脂质化合物(见图1)。
      从TAG标准的位置判断,用铅笔概述TAG点 注意:为防止TAG斑点扩散,应避免喷洒过量的检测试剂。
    9. 通过微刮刀的平坦边缘去掉TAG斑点的硅胶。应立即处理来自TAG的硅胶以生产脂肪酸甲酯(FAME,见下文)以防止其组分脂肪酸的氧化。


      图1. TLC在总脂质组分中的TLC分离。 kessleri

  3. 将构成的脂肪酸转化成脂肪酸甲酯
    1. 取50μl花生酸溶液作为内标(IS,参见配方),用带有PTFE衬里的酚醛盖的玻璃螺旋盖试管(14ml vol。),在N 2 /天然气。
      注意:由于Chlorella kessrelli 11 h没有内源性C20系列脂肪酸,作者使用花生酸(C20:0)作为IS。十九烷酸(C19:0)也可以用作IS。
    2. 将TAG和总脂质部分的5%刮下的硅胶分别放入试管中。后者的脂质溶液必须在N 2气流下风干。剩余的总脂质含量,即初始含量的50%,可以保存在-80°C以备以后分析。
    3. 加入2.0ml的5-10%(w / v)氯化氢 - 甲醇试剂到每个试管中,密封,用涡旋混合器以最大速度搅拌30秒。
    4. 将每个试管在95°C加热2小时,以使TAG脂肪酸或总脂质的甲基酯化。
    5. 将管冷却至室温,并向其中加入2毫升正己烷。用涡旋混合器搅拌管子30秒,以最大速度提取FAME。
    6. 立管1分钟,将其内容物分离成上层(正己烷)和下层(甲醇)层。
    7. 通过玻璃巴斯德移液管将上层相转移到带有PTFE衬里的酚醛盖的玻璃螺旋盖离心管(50ml体积)中。
    8. 加入2.0毫升正己烷到剩余的下层相中并涡旋30秒。
      在室温下将试管放置1分钟

    9. 在步骤C7将上层的相回收到玻璃管中。
    10. 重复步骤C8到C9两次,以强化提取FAME。
    11. 用旋转蒸发器蒸发FAMEs溶液,并将干燥的样品溶解在少量(<50μl)正己烷中。将浓缩的FAMEs溶液转移到2毫升玻璃瓶中的0.15毫升插入物中,然后盖上4℃保存直至测量。
    12. 用毛细管GLC分析样品的脂肪酸组成(见图2)。 GLC按照制造商的说明进行操作。 GLC的操作条件如表1所示。


      图2.来自C. kessleri的TAG的FAME的气相色谱图

      表1.用于测定脂肪酸组成的GLC操作条件

      注意:FID检测通过燃烧碳化合物而产生的离子(Holm,1999)。

数据分析

  1. 根据制造商的说明,用积分仪识别和计算色谱图上各个FAME的峰面积。与标准FAME混合物(Supelco 37组分FAME混合物,用正己烷稀释的100μg/ ml溶液)相比,FAME的鉴定基于保留时间。
    检查色谱图是否与正在显示的适当基线正确集成
  2. 通过使用以下式[1]和花生酸(20:0)作为IS来定量每种FAME的分子种类。值160和312分别表示在步骤C1中得到的20:0(nmol)的含量和20:0的分子量。总脂质的摩尔含量可以基于包含的脂肪酸(式[2])表示。
  3. TAG含量估计为其构成脂肪酸总摩尔含量的1/3(式[3])。请注意,获得的值与分析的各个样本中包含的值相对应。或者,基于脂肪酸估计TAG相对于总脂质的含量(式[4])。如此处所示,根据用于TAG分析的总脂质比例(45%)与总脂质分析(5%)的比例,需要将“来自TAG的FAME的总含量”乘以1/9。定量结果应该用至少三次实验的平均值±标准偏差来表示。



    注:FAME的峰面积由FA的分子量方便地分开,但是除FAME以外的FAME碳原子完全反应(Ackman和Sipos,1964)。




的食谱

  1. 1 / 4GB培养基,含或不含0.6M山梨糖醇
    1. 将Gamborg的B5中等盐混合物(3.3g /包,表2)溶于蒸馏水中以制备正常的Gamborg's B5培养基(GB;最终体积,1L)

      表2. Gamborg B5中等盐混合物的化学组成


    2. 添加750毫升的蒸馏水到250毫升的国标准备1/4 GB
    3. 否则,将10g山梨糖醇溶于250ml GB中,同时加入蒸馏水以制备含有1 / 4GB(最终体积,1L)的0.6M山梨糖醇。
    4. 含1 / 4GB的1/4 GB和0.6M山梨醇必须进行高压灭菌(120°C,20分钟)
  2. 花生酸溶液作为IS
    1. 10毫克的花生四烯酸溶于10毫升的2-丙醇
    2. 将溶液储存在2-8°C

的致谢

这项工作得到了东京药物和生命科学大学的支持。作者宣称不存在相互竞争的利益。

<强>参考

  1. ,R.G.和Sipos,J.C。(1964)。 使用火焰离子化检测器在脂肪酸甲酯的气相色谱分析中应用特定的响应因子。 J Am Oil Chem Soc 41(5):377-378。
  2. Bligh,E.G。和Dyer,W.J。(1959)。 一种快速的总脂质提取和纯化方法可将J Biochem Physiol 37(8):911-917。
  3. Dorschel,C.A。(2002)。 通过电喷雾LC-MS-MS鉴定花生油的TAG。 J美国石油化学家协会(J American Oil Chemists'Society)79(8):749-753。
  4. Gamborg,O. L.,Miller,R.A。和Ojima,K。(1968)。 大豆根细胞悬浮培养的营养需求 > Cell Res 50(1):151-158。
  5. Hayashi,T.,Otaki,R.,Hirai,K.,Tsuzuki,M.and Sato,N.(2017)。 淡水绿藻中基于海水的三酰甘油积累的优化,<小球藻> >,通过同时施加低温和增强光强度。
  6. Hirai,K.,Hayashi,T.,Hasegawa,Y.,Sato,A.,Tsuzuki,M.and Sato,N。(2016)。 高渗及其与营养限制的结合是新型环境应激物,用于诱导三酰甘油在<小球藻。 Sci Rep 6:25825.
  7. Holm T.(1999)。 火焰离子化检测器机理的几个方面 842(1-2):221-227。
  8. MacDougall,K.M.,McNichol,J.,McGinn P.J.,O'Leary,S.J.B。和Melanson,J.E。(2011)。 利用液相色谱 - 高分辨质谱对生物燃料原料的微藻菌株进行三酰甘油分析spectrometry。 Anal Bioanal Chem 401(8):2609-2616。
  9. McGowan,M. W.,Artiss,J. D.,Strandbergh,D. R.和Zak,B。(1983)。 过氧化酶偶合法测定血清甘油三酯的比色法 Chem 29(3):538-542。
  10. Mendez,A.J.,Cabeza,C。和Hsia,S.L。(1986)。 测定纳摩尔量的甘油三酯的荧光测定方法 Anal Biochem 156(2):386-389。
  11. Mu,H.,Sillen,H.和Hiy,C.E。(2000)。 通过常压化学电离液相色谱法鉴定结构化脂质样品中的二酰甘油和三酰甘油/质谱法。 <美国石油化学家协会 77(10):1049-1060。
  12. Sato,N。和Tsuzuki,M。(2011)。 叶绿体脂质的分离与鉴定 Methods Mol Biol 684:95-104。
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
引用:Aoki, M. and Sato, N. (2018). Fatty Acid Content and Composition of Triacylglycerols of Chlorella kessleri. Bio-protocol 8(1): e2676. DOI: 10.21769/BioProtoc.2676.
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